Modulation of transthyretin expression

ABSTRACT

Provided herein are methods, compounds, and compositions for reducing expression of transthyretin mRNA and protein in an animal. Such methods, compounds, and compositions are useful to treat, prevent, delay, or ameliorate transthyretin amyloidosis, or a symptom thereof.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledBIOL0123USC4SEQ_ST25.txt created Dec. 21, 2018 which is 64 Kb in size.The information in the electronic format of the sequence listing isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

Provided herein are methods, compounds, and compositions for reducingexpression of transthyretin mRNA and protein in an animal. Such methods,compounds, and compositions are useful, for example, to treat, prevent,or ameliorate transthyretin amyloidosis.

BACKGROUND OF THE INVENTION

Transthyretin (TTR), (also known as prealbumin, hyperthytoxinemia,dysprealbuminemic, thyroxine; senile systemic amyloidosis, amyloidpolyneuropathy, amyloidosis I, PALB; dystransthyretinemic, HST2651;TBPA; dysprealbuminemic euthyroidal hyperthyroxinemia) is a serum/plasmaand cerebrospinal fluid protein responsible for the transport ofthyroxine and retinol (Sakaki et al, Mol Biol Med. 1989, 6:161-8).Structurally, TTR is a homotetramer; point mutations and misfolding ofthe protein leads to deposition of amyloid fibrils and is associatedwith disorders, such as senile systemic amyloidosis (SSA), familialamyloid polyneuropathy (FAP), and familial amyloid cardiopathy (FAC).

TTR is synthesized primarily by the liver and the choroid plexus of thebrain and, to a lesser degree, by the retina in humans (Palha, Clin ChemLab Med, 2002, 40, 1292-1300). Transthyretin that is synthesized in theliver is secreted into the blood, whereas transthyretin originating inthe choroid plexus is destined for the CSF. In the choroid plexus,transthyretin synthesis represents about 20% of total local proteinsynthesis and as much as 25% of the total CSF protein (Dickson et al., JBiol Chem, 1986, 261, 3475-3478).

With the availability of genetic and immunohistochemical diagnostictests, patients with TTR amyloidosis have been found in many nationsworldwide. Recent studies indicate that TTR amyloidosis is not a rareendemic disease as previously thought, and may affect as much as 25% ofthe elderly population (Tanskanen et al, Ann Med. 2008;40(3):232-9).

At the biochemical level, TTR was identified as the major proteincomponent in the amyloid deposits of FAP patients (Costa et al, Proc.Natl. Acad. Sci. USA 1978, 75:4499-4503) and later, a substitution ofmethionine for valine at position 30 of the protein was found to be themost common molecular defect causing the disease (Saraiva et al, J.Clin. Invest. 1984, 74: 104-119). In FAP, widespread systemicextracellular deposition of TTR aggregates and amyloid fibrils occursthroughout the connective tissue, particularly in the peripheral nervoussystem (Sousa and Saraiva, Prog. Neurobiol. 2003, 71: 385-400).Following TTR deposition, axonal degeneration occurs, starting in theunmyelinated and myelinated fibers of low diameter, and ultimatelyleading to neuronal loss at ganglionic sites.

The compounds and treatment methods described herein provide significantadvantages over the treatments options currently available for TTRrelated disorders. TTR amyloidosis typically lead to death within tenyears, and until recently, was considered incurable. Livertransplantation is an effective means of replacing thedisease-associated allele by a wild type (WT) allele in familial casesbecause the liver is typically the source of amyloidogenic TTR. Whileliver transplantation is effective as a form of gene therapy it is notwithout its problems. Transplantation is complicated by the need forinvasive surgery for the recipient and the donor, long-termpost-transplantation immunosuppressive therapy, a shortage of donors,its high cost, and the large number of TTR amyloidosis patients that arenot good candidates because of their disease progression. Unfortunately,cardiac amyloidosis progresses in some familial patients even afterliver transplantation because WT TTR often continues to deposit. Centralnervous system (CNS) deposition of TTR is also not relieved bytransplantation owing to its synthesis by the choroid plexus.Transplantation is not a viable option for the most prevalent TTRdisease, senile systemic amyloidosis (SSA), affecting approximately 25%of those over 80 due to the deposition of WT TTR.

Antisense technology is emerging as an effective means for reducing theexpression of specific gene products and may therefore prove to beuniquely useful in a number of therapeutic, diagnostic, and researchapplications for the modulation of TTR expression (See U.S. PatentPublication Nos. 2008/0039418 and 2007/0299027.

The present invention provides compositions and methods for modulatingtransthyretin expression. Antisense compounds for modulating expressionof transthyretin are disclosed in the aforementioned published patentapplications. However, there remains a need for additional suchcompounds.

SUMMARY OF THE INVENTION

Provided herein are methods, compounds, and compositions for modulatingexpression of transthyretin (TTR) mRNA and protein. In certainembodiments, compounds useful for modulating expression of TTR mRNA andprotein are antisense compounds. In certain embodiments, the antisensecompounds are antisense oligonucleotides.

In certain embodiments, modulation can occur in a cell or tissue. Incertain embodiments, the cell or tissue is in an animal. In certainembodiments, the animal is a human. In certain embodiments, TTR mRNAlevels are reduced. In certain embodiments, TTR protein levels arereduced. Such reduction can occur in a time-dependent manner or in adose-dependent manner.

Provided herein are methods, compounds, and compositions for modulatingexpression of transthyretin and treating, preventing, delaying orameliorating transthyretin amyloidosis and or a symptom thereof. Incertain embodiments are methods, compounds, and compositions formodulating expression of transthyretin and treating, preventing,delaying or ameliorating transthyretin amyloid disease or transthyretinamyloidosis or transthyretin related amyloidosis (e.g., hereditary TTRamyloidosis, leptomeningeal amyloidosis, transthyretin amyloidpolyneuropathy, familial amyloid polyneuropathy, familial amyloidcardiomyopathy, or senile systemic amyloidosis).

In certain embodiments, an animal at risk for transthyretin amyloidosisis treated by administering to the animal a therapeutically effectiveamount of a compound comprising a modified oligonucleotide consisting of8 to 80 linked nucleosides, wherein the modified oligonucleotide iscomplementary to a transthyretin nucleic acid as shown in SEQ ID NO: 1or SEQ ID NO: 2; or a therapeutically effective amount of a compoundcomprising a modified oligonucleotide consisting of 8 to 80 linkednucleosides and having a nucleobase sequence comprising at least 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 contiguous nucleobases of anucleobase sequence selected from any one of nucleobase sequencesrecited in SEQ ID NOs: 25, 80, 86, 87, 115, 120, 122, or 124.

In certain embodiments, an animal at risk for transthyretin amyloidosisis treated by administering to the animal a therapeutically effectiveamount of a compound comprising a modified oligonucleotide consisting of12 to 50 linked nucleosides, wherein the modified oligonucleotide iscomplementary to a transthyretin nucleic acid as shown in SEQ ID NO: 1or SEQ ID NO: 2; or a therapeutically effective amount of a compoundcomprising a modified oligonucleotide consisting of 12 to 50 linkednucleosides and having a nucleobase sequence comprising at least 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 contiguous nucleobases of anucleobase sequence selected from any one of nucleobase sequencesrecited in SEQ ID NOs: 25, 80, 86, 87, 115, 120, 122, or 124.

In certain embodiments, an animal at risk for transthyretin amyloidosisis treated by administering to the animal a therapeutically effectiveamount of a compound comprising a modified oligonucleotide consisting of12 to 30 linked nucleosides, wherein the modified oligonucleotide iscomplementary to a transthyretin nucleic acid as shown in SEQ ID NO: 1or SEQ ID NO: 2; or a therapeutically effective amount of a compoundcomprising a modified oligonucleotide consisting of 12 to 30 linkednucleosides and having a nucleobase sequence comprising at least 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 contiguous nucleobases of anucleobase sequence selected from any one of nucleobase sequencesrecited in SEQ ID NOs: 25, 80, 86, 87, 115, 120, 122, or 124.

In certain embodiments, an animal at risk for transthyretin amyloidosisis treated by administering to the animal a therapeutically effectiveamount of a compound comprising a modified oligonucleotide consisting of15 to 25 linked nucleosides, wherein the modified oligonucleotide iscomplementary to a transthyretin nucleic acid as shown in SEQ ID NO: 1or SEQ ID NO: 2; or a therapeutically effective amount of a compoundcomprising a modified oligonucleotide consisting of 15 to 25 linkednucleosides and having a nucleobase sequence comprising at least 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 contiguous nucleobases of anucleobase sequence selected from any one of nucleobase sequencesrecited in SEQ ID NOs: 25, 80, 86, 87, 115, 120, 122, or 124.

In certain embodiments, an animal at risk for transthyretin amyloidosisis treated by administering to the animal a therapeutically effectiveamount of a compound comprising a modified oligonucleotide consisting of18 to 21 linked nucleosides, wherein the modified oligonucleotide iscomplementary to a transthyretin nucleic acid as shown in SEQ ID NO: 1or SEQ ID NO: 2; or a therapeutically effective amount of a compoundcomprising a modified oligonucleotide consisting of 18 to 21 linkednucleosides and having a nucleobase sequence comprising at least 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 contiguous nucleobases of anucleobase sequence selected from any one of nucleobase sequencesrecited in SEQ ID NOs: 25, 80, 86, 87, 115, 120, 122, or 124.

In certain embodiments, an animal at risk for transthyretin amyloidosisis treated by administering to the animal a therapeutically effectiveamount of a compound comprising a modified oligonucleotide consisting of20 to 30 linked nucleosides, wherein the modified oligonucleotide iscomplementary to a transthyretin nucleic acid as shown in SEQ ID NO: 1or SEQ ID NO: 2; or a therapeutically effective amount of a compoundcomprising a modified oligonucleotide consisting of 20 to 30 linkednucleosides and having a nucleobase sequence comprising at least 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 contiguous nucleobases of anucleobase sequence selected from any one of nucleobase sequencesrecited in SEQ ID NOs: 25, 80, 86, 87, 115, 120, 122, or 124.

In certain embodiments, an animal having transthyretin amyloidosis istreated by administering to the animal a therapeutically effectiveamount of a compound comprising a modified oligonucleotide consisting of8 to 80 linked nucleosides, wherein the modified oligonucleotide iscomplementary to a transthyretin nucleic acid as shown in SEQ ID NO: 1or SEQ ID NO: 2, or a therapeutically effective amount of a compoundcomprising a modified oligonucleotide consisting of 8 to 80 linkednucleosides and having a nucleobase sequence comprising at least 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 contiguous nucleobases of anucleobase sequence selected from any one of nucleobase sequencesrecited in SEQ ID NOs: 25, 80, 86, 87, 115, 120, 122, or 124.

In certain embodiments, an animal having transthyretin amyloidosis istreated by administering to the animal a therapeutically effectiveamount of a compound comprising a modified oligonucleotide consisting of12 to 50 linked nucleosides, wherein the modified oligonucleotide iscomplementary to a transthyretin nucleic acid as shown in SEQ ID NO: 1or SEQ ID NO: 2, or a therapeutically effective amount of a compoundcomprising a modified oligonucleotide consisting of 12 to 50 linkednucleosides and having a nucleobase sequence comprising at least 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 contiguous nucleobases of anucleobase sequence selected from any one of nucleobase sequencesrecited in SEQ ID NOs: 25, 80, 86, 87, 115, 120, 122, or 124.

In certain embodiments, an animal having transthyretin amyloidosis istreated by administering to the animal a therapeutically effectiveamount of a compound comprising a modified oligonucleotide consisting of12 to 30 linked nucleosides, wherein the modified oligonucleotide iscomplementary to a transthyretin nucleic acid as shown in SEQ ID NO: 1or SEQ ID NO: 2, or a therapeutically effective amount of a compoundcomprising a modified oligonucleotide consisting of 12 to 30 linkednucleosides and having a nucleobase sequence comprising at least 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 contiguous nucleobases of anucleobase sequence selected from any one of nucleobase sequencesrecited in SEQ ID NOs: 25, 80, 86, 87, 115, 120, 122, or 124.

In certain embodiments, an animal having transthyretin amyloidosis istreated by administering to the animal a therapeutically effectiveamount of a compound comprising a modified oligonucleotide consisting of15 to 25 linked nucleosides, wherein the modified oligonucleotide iscomplementary to a transthyretin nucleic acid as shown in SEQ ID NO: 1or SEQ ID NO: 2; or a therapeutically effective amount of a compoundcomprising a modified oligonucleotide consisting of 15 to 25 linkednucleosides and having a nucleobase sequence comprising at least 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 contiguous nucleobases of anucleobase sequence selected from any one of nucleobase sequencesrecited in SEQ ID NOs: 25, 80, 86, 87, 115, 120, 122, or 124.

In certain embodiments, an animal having transthyretin amyloidosis istreated by administering to the animal a therapeutically effectiveamount of a compound comprising a modified oligonucleotide consisting of18 to 21 linked nucleosides, wherein the modified oligonucleotide iscomplementary to a transthyretin nucleic acid as shown in SEQ ID NO: 1or SEQ ID NO: 2; or a therapeutically effective amount of a compoundcomprising a modified oligonucleotide consisting of 18 to 21 linkednucleosides and having a nucleobase sequence comprising at least 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 contiguous nucleobases of anucleobase sequence selected from any one of nucleobase sequencesrecited in SEQ ID NOs: 25, 80, 86, 87, 115, 120, 122, or 124.

In certain embodiments, an animal at risk for transthyretin amyloidosisor having transthyretin amyloidosis is treated by administering to theanimal a therapeutically effective amount of a compound comprising amodified oligonucleotide consisting of 8 to 80 linked nucleosides,wherein the modified oligonucleotide is complementary to a transthyretinnucleic acid as shown in SEQ ID NO: 1 or SEQ ID NO: 2; or atherapeutically effective amount of a compound comprising a modifiedoligonucleotide consisting of 8 to 80 linked nucleosides and having anucleobase sequence comprising at least 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19 or 20 contiguous nucleobases of a nucleobase sequenceselected from any one of nucleobase sequences recited in SEQ ID NOs: 25,80, 86, or 87.

In certain embodiments, an animal at risk for transthyretin amyloidosisor having transthyretin amyloidosis is treated by administering to theanimal a therapeutically effective amount of a compound comprising amodified oligonucleotide consisting of 12 to 50 linked nucleosides,wherein the modified oligonucleotide is complementary to a transthyretinnucleic acid as shown in SEQ ID NO: 1 or SEQ ID NO: 2; or atherapeutically effective amount of a compound comprising a modifiedoligonucleotide consisting of 12 to 50 linked nucleosides and having anucleobase sequence comprising at least 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19 or 20 contiguous nucleobases of a nucleobase sequenceselected from any one of nucleobase sequences recited in SEQ ID NOs: 25,80, 86, or 87.

In certain embodiments, an animal at risk for transthyretin amyloidosisor having transthyretin amyloidosis is treated by administering to theanimal a therapeutically effective amount of a compound comprising amodified oligonucleotide consisting of 12 to 30 linked nucleosides,wherein the modified oligonucleotide is complementary to a transthyretinnucleic acid as shown in SEQ ID NO: 1 or SEQ ID NO: 2; or atherapeutically effective amount of a compound comprising a modifiedoligonucleotide consisting of 12 to 30 linked nucleosides and having anucleobase sequence comprising at least 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19 or 20 contiguous nucleobases of a nucleobase sequenceselected from any one of nucleobase sequences recited in SEQ ID NOs: 25,80, 86, or 87.

In certain embodiments, an animal at risk for transthyretin amyloidosisor having transthyretin amyloidosis is treated by administering to theanimal a therapeutically effective amount of a compound comprising amodified oligonucleotide consisting of 15 to 25 linked nucleosides,wherein the modified oligonucleotide is complementary to a transthyretinnucleic acid as shown in SEQ ID NO: 1 or SEQ ID NO: 2; or atherapeutically effective amount of a compound comprising a modifiedoligonucleotide consisting of 15 to 25 linked nucleosides and having anucleobase sequence comprising at least 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19 or 20 contiguous nucleobases of a nucleobase sequenceselected from any one of nucleobase sequences recited in SEQ ID NOs: 25,80, 86, or 87.

In certain embodiments, an animal at risk for transthyretin amyloidosisor having transthyretin amyloidosis is treated by administering to theanimal a therapeutically effective amount of a compound comprising amodified oligonucleotide consisting of 18 to 21 linked nucleosides,wherein the modified oligonucleotide is complementary to a transthyretinnucleic acid as shown in SEQ ID NO: 1 or SEQ ID NO: 2; or atherapeutically effective amount of a compound comprising a modifiedoligonucleotide consisting of 18 to 21 linked nucleosides and having anucleobase sequence comprising at least 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19 or 20 contiguous nucleobases of a nucleobase sequenceselected from any one of nucleobase sequences recited in SEQ ID NOs: 25,80, 86, or 87.

In certain embodiments, an animal at risk for transthyretin amyloidosisor having transthyretin amyloidosis is treated by administering to theanimal a therapeutically effective amount of a compound comprising amodified oligonucleotide consisting of 20 to 30 linked nucleosides,wherein the modified oligonucleotide is complementary to a transthyretinnucleic acid as shown in SEQ ID NO: 1 or SEQ ID NO: 2; or atherapeutically effective amount of a compound comprising a modifiedoligonucleotide consisting of 20 to 30 linked nucleosides and having anucleobase sequence comprising at least 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19 or 20 contiguous nucleobases of a nucleobase sequenceselected from any one of nucleobase sequences recited in SEQ ID NOs: 25,80, 86, or 87.

In certain embodiments, an animal at risk for transthyretin amyloidosisor having transthyretin amyloidosis is treated by administering to theanimal a therapeutically effective amount of a compound comprising amodified oligonucleotide consisting of 8 to 80 linked nucleosides,wherein the modified oligonucleotide is complementary to a transthyretinnucleic acid as shown in SEQ ID NO: 1 or SEQ ID NO: 2; or atherapeutically effective amount of a compound comprising a modifiedoligonucleotide consisting of 8 to 80 linked nucleosides and having anucleobase sequence comprising at least 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19 or 20 contiguous nucleobases of the nucleobase sequencerecited in SEQ ID NO: 80.

In certain embodiments, an animal at risk for transthyretin amyloidosisor having transthyretin amyloidosis is treated by administering to theanimal a therapeutically effective amount of a compound comprising amodified oligonucleotide consisting of 12 to 50 linked nucleosides,wherein the modified oligonucleotide is complementary to a transthyretinnucleic acid as shown in SEQ ID NO: 1 or SEQ ID NO: 2; or atherapeutically effective amount of a compound comprising a modifiedoligonucleotide consisting of 12 to 50 linked nucleosides and having anucleobase sequence comprising at least 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19 or 20 contiguous nucleobases of the nucleobase sequencerecited in SEQ ID NO: 80.

In certain embodiments, an animal at risk for transthyretin amyloidosisor having transthyretin amyloidosis is treated by administering to theanimal a therapeutically effective amount of a compound comprising amodified oligonucleotide consisting of 12 to 30 linked nucleosides,wherein the modified oligonucleotide is complementary to a transthyretinnucleic acid as shown in SEQ ID NO: 1 or SEQ ID NO: 2; or atherapeutically effective amount of a compound comprising a modifiedoligonucleotide consisting of 12 to 30 linked nucleosides and having anucleobase sequence comprising at least 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19 or 20 contiguous nucleobases of the nucleobase sequencerecited in SEQ ID NO: 80.

In certain embodiments, an animal at risk for transthyretin amyloidosisor having transthyretin amyloidosis is treated by administering to theanimal a therapeutically effective amount of a compound comprising amodified oligonucleotide consisting of 15 to 25 linked nucleosides,wherein the modified oligonucleotide is complementary to a transthyretinnucleic acid as shown in SEQ ID NO: 1 or SEQ ID NO: 2; or atherapeutically effective amount of a compound comprising a modifiedoligonucleotide consisting of 15 to 25 linked nucleosides and having anucleobase sequence comprising at least 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19 or 20 contiguous nucleobases of the nucleobase sequencerecited in SEQ ID NO: 80.

In certain embodiments, an animal at risk for transthyretin amyloidosisor having transthyretin amyloidosis is treated by administering to theanimal a therapeutically effective amount of a compound comprising amodified oligonucleotide consisting of 18 to 21 linked nucleosides,wherein the modified oligonucleotide is complementary to a transthyretinnucleic acid as shown in SEQ ID NO: 1 or SEQ ID NO: 2; or atherapeutically effective amount of a compound comprising a modifiedoligonucleotide consisting of 18 to 21 linked nucleosides and having anucleobase sequence comprising at least 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19 or 20 contiguous nucleobases of the nucleobase sequencerecited in SEQ ID NO: 80.

In certain embodiments, an animal at risk for transthyretin amyloidosisor having transthyretin amyloidosis is treated by administering to theanimal a therapeutically effective amount of a compound comprising amodified oligonucleotide consisting of 20 to 30 linked nucleosides,wherein the modified oligonucleotide is complementary to a transthyretinnucleic acid as shown in SEQ ID NO: 1 or SEQ ID NO: 2; or atherapeutically effective amount of a compound comprising a modifiedoligonucleotide consisting of 20 to 30 linked nucleosides and having anucleobase sequence comprising at least 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19 or 20 contiguous nucleobases of the nucleobase sequencerecited in SEQ ID NO: 80.

In certain embodiments, an animal at risk for transthyretin amyloidosisor having transthyretin amyloidosis is treated by administering to theanimal a therapeutically effective amount of a compound comprising amodified oligonucleotide consisting of 20 linked nucleosides, whereinthe modified oligonucleotide is complementary to a transthyretin nucleicacid as shown in SEQ ID NO: 1 or SEQ ID NO: 2; or a therapeuticallyeffective amount of a compound comprising a modified oligonucleotideconsisting of 20 linked nucleosides and having a nucleobase sequencecomprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20contiguous nucleobases of the nucleobase sequence recited in SEQ ID NO:80.

In certain embodiments, an animal having transthyretin amyloidosis istreated by administering to the animal a therapeutically effectiveamount of a compound comprising a modified oligonucleotide consisting of20 linked nucleosides, wherein the modified oligonucleotide is 100%complementary to a transthyretin nucleic acid as shown in SEQ ID NO: 1or SEQ ID NO: 2; or a therapeutically effective amount of a compoundcomprising a modified oligonucleotide consisting of 20 linkednucleosides and having the nucleobase sequence recited in SEQ ID NO: 80.

In certain embodiments, an animal having transthyretin amyloidosis istreated by administering to the animal a therapeutically effectiveamount of a compound comprising a modified oligonucleotide consisting of20 linked nucleosides, wherein the modified oligonucleotide is 100%complementary to a transthyretin nucleic acid as shown in SEQ ID NO: 1;and wherein the compound comprises a modified oligonucleotide consistingof 20 linked nucleosides having the nucleobase sequence recited in SEQID NO: 80.

In certain embodiments, an animal having transthyretin amyloidosis istreated by administering to the animal a therapeutically effectiveamount of a compound comprising a modified oligonucleotide consisting of20 linked nucleosides, wherein the modified oligonucleotide is 100%complementary to a transthyretin nucleic acid as shown in SEQ ID NO: 1;wherein the compound comprises a modified oligonucleotide consisting of20 linked nucleosides having the nucleobase sequence recited in SEQ IDNO: 80; and wherein the modified oligonucleotides has a gap segment of10 linked deoxynucleosides between two wing segments that independentlyhave 5 linked modified nucleosides. In certain embodiments, one or moremodified nucleosides in the wing segment have a modified sugar. Incertain embodiments, the modified nucleoside is a 2′-substitutednucleoside. In certain embodiments, the modified nucleoside is a 2′-MOEnucleoside.

In certain embodiments, modulation can occur in a cell, tissue, organ ororganism. In certain embodiments, the cell, tissue or organ is in ananimal. In certain embodiments, the animal is a human. In certainembodiments, transthyretin mRNA levels are reduced. In certainembodiments, transthyretin protein levels are reduced. Such reductioncan occur in a time-dependent manner or in a dose-dependent manner.

Also provided are methods, compounds, and compositions useful forpreventing, treating, and ameliorating diseases, disorders, andconditions related to transthyretin amyloidosis. In certain embodiments,such diseases, disorders, and conditions are transthyretin amyloidosisrelated diseases disorders or conditions.

In certain embodiments, methods of treatment include administering a TTRantisense compound to an individual in need thereof. In certainembodiments, methods of treatment include administering a TTR antisenseoligonucleotide to an individual in need thereof

In certain embodiments, methods of treatment include administering atransthyretin antisense oligonucleotide and an additional therapy to anindividual in need thereof.

DETAILED DESCRIPTION OF THE INVENTION

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention, as claimed. Herein, the use ofthe singular includes the plural unless specifically stated otherwise.As used herein, the use of “or” means “and/or” unless stated otherwise.Furthermore, the use of the term “including” as well as other forms,such as “includes” and “included”, is not limiting. Also, terms such as“element” or “component” encompass both elements and componentscomprising one unit and elements and components that comprise more thanone subunit, unless specifically stated otherwise.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All documents, or portions of documents, cited in this application,including, but not limited to, patents, patent applications, articles,books, and treatises, are hereby expressly incorporated by reference forthe portions of the document discussed herein, as well as in theirentirety.

Definitions

Unless specific definitions are provided, the nomenclature utilized inconnection with, and the procedures and techniques of, analyticalchemistry, synthetic organic chemistry, and medicinal and pharmaceuticalchemistry described herein are those well known and commonly used in theart. Standard techniques may be used for chemical synthesis, andchemical analysis. Where permitted, all patents, applications, publishedapplications and other publications, GENBANK Accession Numbers andassociated sequence information obtainable through databases such asNational Center for Biotechnology Information (NCBI) and other datareferred to throughout in the disclosure herein are incorporated byreference for the portions of the document discussed herein, as well asin their entirety.

Unless otherwise indicated, the following terms have the followingmeanings:

“2′—O-methoxyethyl” (also 2′-MOE and 2′—O(CH₂)₂—OCH₃) refers to anO-methoxy-ethyl modification of the 2′ position of a furosyl ring. A2′—O-methoxyethyl modified sugar is a modified sugar.

“2′—O-methoxyethyl nucleotide” means a nucleotide comprising a2′—O-methoxyethyl modified sugar moiety.

“5-methylcytosine” means a cytosine modified with a methyl groupattached to the 5′ position. A 5-methylcytosine is a modifiednucleobase.

“Active pharmaceutical agent” means the substance or substances in apharmaceutical composition that provide a therapeutic benefit whenadministered to an individual. For example, in certain embodiments anantisense oligonucleotide targeted to transthyretin is an activepharmaceutical agent.

“Active target region” or “target region” means a region to which one ormore active antisense compounds is targeted. “Active antisensecompounds” means antisense compounds that reduce target nucleic acidlevels or protein levels.

“Administered concomitantly” refers to the co-administration of twoagents in any manner in which the pharmacological effects of both aremanifest in the patient at the same time. Concomitant administrationdoes not require that both agents be administered in a singlepharmaceutical composition, in the same dosage form, or by the sameroute of administration. The effects of both agents need not manifestthemselves at the same time. The effects need only be overlapping for aperiod of time and need not be coextensive.

“Administering” means providing a pharmaceutical agent to an individual,and includes, but is not limited to administering by a medicalprofessional and self-administering.

“Amelioration” refers to a lessening of at least one indicator, sign, orsymptom of an associated disease, disorder, or condition. The severityof indicators may be determined by subjective or objective measures,which are known to those skilled in the art.

“Amyloidosis” is a group of diseases or disorders resulting fromabnormal protein (amyloid or amyloid fibril) deposits in various bodytissues. The amyloid proteins may either be deposited in one particulararea of the body (localized amyloidosis) or they may be depositedthroughout the body (systemic amyloidosis). There are three types ofsystemic amyloidosis: primary (AL), secondary (AA), and familial (ATTR).Primary amyloidosis is not associated with any other diseases and isconsidered a disease entity of its own. Secondary amyloidosis occurs asa result of another illness. Familial Mediterranean Fever is a form offamilial (inherited) amyloidosis.

“Animal” refers to a human or non-human animal, including, but notlimited to, mice, rats, rabbits, dogs, cats, pigs, and non-humanprimates, including, but not limited to, monkeys and chimpanzees.

“Antisense activity” means any detectable or measurable activityattributable to the hybridization of an antisense compound to its targetnucleic acid. In certain embodiments, antisense activity is a decreasein the amount or expression of a target nucleic acid or protein encodedby such target nucleic acid.

“Antisense compound” means an oligomeric compound that is capable ofundergoing hybridization to a target nucleic acid through hydrogenbonding.

“Antisense inhibition” means reduction of target nucleic acid levels ortarget protein levels in the presence of an antisense compoundcomplementary to a target nucleic acid compared to target nucleic acidlevels or target protein levels in the absence of the antisensecompound.

“Antisense oligonucleotide” means a single-stranded oligonucleotidehaving a nucleobase sequence that permits hybridization to acorresponding region or segment of a target nucleic acid.

“Bicyclic sugar” means a furosyl ring modified by the bridging of twonon-geminal ring atoms. A bicyclic sugar is a modified sugar.

“Bicyclic nucleic acid” or “BNA” refers to a nucleoside or nucleotidewherein the furanose portion of the nucleoside or nucleotide includes abridge connecting two carbon atoms on the furanose ring, thereby forminga bicyclic ring system.

“Cap structure” or “terminal cap moiety” means chemical modifications,which have been incorporated at either terminus of an antisensecompound.

“Central nervous system (CNS)” refers to the vertebrate nervous systemwhich is enclosed in meninges. It contains the majority of the nervoussystem, and consists of the brain (in vertebrates which have brains),and the spinal cord. The CNS is contained within the dorsal cavity, withthe brain within the cranial cavity, and the spinal cord in the spinalcavity. The brain is also protected by the skull, and the spinal cordis, in vertebrates, also protected by the vertebrae.

“Chemically distinct region” refers to a region of an antisense compoundthat is in some way chemically different than another region of the sameantisense compound. For example, a region having 2′—O-methoxyethylnucleotides is chemically distinct from a region having nucleotideswithout 2′—O-methoxyethyl modifications.

“Chimeric antisense compound” means an antisense compound that has atleast two chemically distinct regions.

“Choroid plexus” is the area on the ventricles of the brain wherecerebrospinal fluid (CSF) is produced.

“Co-administration” means administration of two or more pharmaceuticalagents to an individual. The two or more pharmaceutical agents may be ina single pharmaceutical composition, or may be in separatepharmaceutical compositions. Each of the two or more pharmaceuticalagents may be administered through the same or different routes ofadministration. Co-administration encompasses parallel or sequentialadministration.

“Complementarity” means the capacity for pairing between nucleobases ofa first nucleic acid and a second nucleic acid.

“Contiguous nucleobases” means nucleobases immediately adjacent to eachother.

“Diluent” means an ingredient in a composition that lackspharmacological activity, but is pharmaceutically necessary ordesirable. For example, the diluent in an injected composition may be aliquid, e.g. saline solution.

“Dose” means a specified quantity of a pharmaceutical agent provided ina single administration, or in a specified time period. In certainembodiments, a dose may be administered in one, two, or more boluses,tablets, or injections. For example, in certain embodiments wheresubcutaneous administration is desired, the desired dose requires avolume not easily accommodated by a single injection, therefore, two ormore injections may be used to achieve the desired dose. In certainembodiments, the pharmaceutical agent is administered by infusion overan extended period of time or continuously. Doses may be stated as theamount of pharmaceutical agent per hour, day, week, or month.

“Effective amount” means the amount of active pharmaceutical agentsufficient to effectuate a desired physiological outcome in anindividual in need of the agent. The effective amount may vary amongindividuals depending on the health and physical condition of theindividual to be treated, the taxonomic group of the individuals to betreated, the formulation of the composition, assessment of theindividual's medical condition, and other relevant factors.

“Familial amyloidosis” or “hereditary amyloidosis” is a form ofinherited amyloidosis.

“Familial amyloid polyneuropathy” or “FAP” is a neurodegenerativegenetically transmitted disorder, characterized by systemic depositionsof amyloid variants of transthyretin proteins, causing progressivesensory and motorial polyneuropathy.

“Fully complementary” or “100% complementary” means each nucleobase of anucleobase sequence of a first nucleic acid has a complementarynucleobase in a second nucleobase sequence of a second nucleic acid. Incertain embodiments, a first nucleic acid is an antisense compound and atarget nucleic acid is a second nucleic acid.

“Gapmer” means a chimeric antisense compound in which an internal regionhaving a plurality of nucleosides that support RNase H cleavage ispositioned between external regions having one or more nucleosides,wherein the nucleosides comprising the internal region are chemicallydistinct from the nucleoside or nucleosides comprising the externalregions. The internal region may be referred to as a “gap segment” andthe external regions may be referred to as “wing segments.”

“Gap-widened” means a chimeric antisense compound having a gap segmentof 12 or more contiguous 2′-deoxyribonucleosides positioned between andimmediately adjacent to 5′ and 3′ wing segments having from one to sixnucleosides.

“Hereditary transthyretin (TTR) amyloidosis” is a systemic diseasecaused by mutations in transthyretin, a plasma transport protein forthyroxine and vitamin A. It is most frequently associated withperipheral neuropathy and restrictive cardiomyopathy, but amyloiddeposits in blood vessel walls and connective tissue structuresthroughout the body often cause dysfunction of other organ systems.Gastrointestinal motility abnormalities are common in this disease withconstipation, diarrhea and early satiety from delayed gastric-emptying.Connective tissue deposits of amyloid in the wrist may cause carpaltunnel syndrome. Amyloid deposits in spinal blood vessels andsurrounding structures cause spinal stenosis with symptoms ofclaudication.

“Hybridization” means the annealing of complementary nucleic acidmolecules. In certain embodiments, complementary nucleic acid moleculesinclude an antisense compound and a target nucleic acid.

“Immediately adjacent” means there are no intervening elements betweenthe immediately adjacent elements.

“Individual” means a human or non-human animal selected for treatment ortherapy.

“Intracerebroventricular administration” or “cerebral intraventricularadministration” or “cerebral ventricular administration” meansadministration through injection or infusion into the ventricular systemof the brain.

“Intraperitoneal administration” means administration to the peritonealcavity.

“Intrathecal administration” means administration through injection orinfusion into the cerebrospinal fluid bathing the spinal cord and brain.

“Intravenous administration” means administration into a vein.

“Intraventricular administration” means administration into theventricles of either the brain or heart.

“Internucleoside linkage” refers to the chemical bond betweennucleosides.

“Leptomeningeal” means having to do with the leptomeninges, the twoinnermost layers of tissues that cover the brain and spinal cord.“Leptomeningeal amyloidosis” refers to amyloidosis of the leptomeningesresulting from transthyretin amyloid deposition within theleptomeninges.

“Linked nucleosides” means adjacent nucleosides which are bondedtogether.

“Mismatch” or “non-complementary nucleobase” refers to the case when anucleobase of a first nucleic acid is not capable of pairing with thecorresponding nucleobase of a second or target nucleic acid.

“Modified internucleoside linkage” refers to a substitution or anychange from a naturally occurring internucleoside bond (i.e. aphosphodiester internucleoside bond).

“Modified nucleobase” refers to any nucleobase other than adenine,cytosine, guanine, thymidine, or uracil. An “unmodified nucleobase”means the purine bases adenine (A) and guanine (G), and the pyrimidinebases thymine (T), cytosine (C), and uracil (U).

“Modified nucleotide” means a nucleotide having, independently, amodified sugar moiety, modified internucleoside linkage, or modifiednucleobase. A “modified nucleoside” means a nucleoside having,independently, a modified sugar moiety or modified nucleobase.

“Modified oligonucleotide” means an oligonucleotide comprising at leastone modified nucleotide.

“Modified sugar” refers to a substitution or change from a naturalsugar.

“Motif” means the pattern of chemically distinct regions in an antisensecompound.

“Naturally occurring internucleoside linkage” means a 3′ to 5′phosphodiester linkage.

“Natural sugar moiety” means a sugar found in DNA (2′—H) or RNA (2′—OH).

“Nucleic acid” refers to molecules composed of monomeric nucleotides. Anucleic acid includes ribonucleic acids (RNA), deoxyribonucleic acids(DNA), single-stranded nucleic acids, double-stranded nucleic acids,small interfering ribonucleic acids (siRNA), and microRNAs (miRNA). Anucleic acid may also comprise a combination of these elements in asingle molecule.

“Nucleobase” means a heterocyclic moiety capable of pairing with a baseof another nucleic acid.

“Nucleobase sequence” means the order of contiguous nucleobasesindependent of any sugar, linkage, or nucleobase modification.

“Nucleoside” means a nucleobase linked to a sugar.

“Nucleotide” means a nucleoside having a phosphate group covalentlylinked to the sugar portion of the nucleoside.

“Oligomeric compound” or “oligomer” means a polymer of linked monomericsubunits which is capable of hybridizing to at least a region of anucleic acid molecule.

“Oligonucleotide” means a polymer of linked nucleosides each of whichcan be modified or unmodified, independent one from another.

“Parenteral administration” means administration through injection orinfusion. Parenteral administration includes subcutaneousadministration, intravenous administration, intramuscularadministration, intraarterial administration, intraperitonealadministration, or intracranial administration, e.g. intracerebraladministration, intrathecal administration, intraventricularadministration, ventricular administration, intracerebroventricularadministration, cerebral intraventricular administration or cerebralventricular administration. Administration can be continuous, orchronic, or short or intermittent.

“Peptide” means a molecule formed by linking at least two amino acids byamide bonds. Peptide refers to polypeptides and proteins.

“Pharmaceutical composition” means a mixture of substances suitable foradministering to an individual. For example, a pharmaceuticalcomposition may comprise one or more active pharmaceutical agents and asterile aqueous solution.

“Pharmaceutically acceptable salts” means physiologically andpharmaceutically acceptable salts of antisense compounds, i.e., saltsthat retain the desired biological activity of the parentoligonucleotide and do not impart undesired toxicological effectsthereto.

“Phosphorothioate linkage” means a linkage between nucleosides where thephosphodiester bond is modified by replacing one of the non-bridgingoxygen atoms with a sulfur atom. A phosphorothioate linkage is amodified internucleoside linkage.

“Portion” means a defined number of contiguous (i.e. linked) nucleobasesof a nucleic acid. In certain embodiments, a portion is a defined numberof contiguous nucleobases of a target nucleic acid. In certainembodiments, a portion is a defined number of contiguous nucleobases ofan antisense compound.

“Prevent” refers to delaying or forestalling the onset or development ofa disease, disorder, or condition for a period of time from minutes toindefinitely. Prevent also means reducing risk of developing a disease,disorder, or condition.

“Prodrug” means a therapeutic agent that is prepared in an inactive formthat is converted to an active form within the body or cells thereof bythe action of endogenous enzymes or other chemicals or conditions.

“Side effects” means physiological responses attributable to a treatmentother than the desired effects. In certain embodiments, side effectsinclude injection site reactions, liver function test abnormalities,renal function abnormalities, liver toxicity, renal toxicity, centralnervous system abnormalities, myopathies, and malaise. For example,increased aminotransferase levels in serum may indicate liver toxicityor liver function abnormality. For example, increased bilirubin mayindicate liver toxicity or liver function abnormality.

“Single-stranded oligonucleotide” means an oligonucleotide which is nothybridized to a complementary strand.

“Specifically hybridizable” refers to an antisense compound having asufficient degree of complementarity between an antisenseoligonucleotide and a target nucleic acid to induce a desired effect,while exhibiting minimal or no effects on non-target nucleic acids underconditions in which specific binding is desired, i.e. underphysiological conditions in the case of in vivo assays and therapeutictreatments.

“Subcutaneous administration” means administration just below the skin.

“Targeting” or “targeted” means the process of design and selection ofan antisense compound that will specifically hybridize to a targetnucleic acid and induce a desired effect.

“Target nucleic acid,” “target RNA,” and “target RNA transcript” allrefer to a nucleic acid capable of being targeted by antisensecompounds.

“Target segment” means the sequence of nucleotides of a target nucleicacid to which an antisense compound is targeted. “5′ target site” refersto the 5′-most nucleotide of a target segment. “3′ target site” refersto the 3′-most nucleotide of a target segment.

“Therapeutically effective amount” means an amount of a pharmaceuticalagent that provides a therapeutic benefit to an individual.

“Transthyretin-specific inhibitor” or “Transthyretin inhibitor” meansany compound capable of decreasing transthyretin mRNA or proteinexpression. Examples of such compounds include a nucleic acid, apeptide, an antibody, or a histone deacetylase inhibitor.

“Transthyretin specific modulator” or “transthyretin modulator” meansany compound capable of increasing or decreasing transthyretin mRNA orprotein expression.

“Transthyretin-related amyloidosis” or “transthyretin amyloidosis” or“Transthyretin amyloid disease”, as used herein, is any pathology ordisease associated with dysfunction or dysregulation of transthyretinthat result in formation of transthyretin-containing amyloid fibrils.Transthyretin amyloidosis includes, but is not limited to, hereditaryTTR amyloidosis, leptomeningeal amyloidosis, familial amyloidpolyneuropathy (FAP), familial amyloid cardiomyopathy, familialoculoleptomeningeal amyloidosis, senile cardiac amyloidosis, or senilesystemic amyloidosis.,

“Treat” refers to administering a pharmaceutical composition to effectan alteration or improvement of a disease, disorder, or condition.

“Unmodified nucleotide” means a nucleotide composed of naturallyoccurring nucleobases, sugar moieties, and internucleoside linkages. Incertain embodiments, an unmodified nucleotide is an RNA nucleotide (i.e.β-D-ribonucleosides) or a DNA nucleotide (i.e. β-D-deoxyribonucleoside).

Certain Embodiments

Certain embodiments provide methods, compounds, and compositions forinhibiting transthyretin expression.

Certain embodiments provide antisense compounds targeted to atransthyretin nucleic acid. In certain embodiments, the transthyretinnucleic acid is any of the sequences set forth in GENBANK Accession No.NM_000371.2 (incorporated herein as SEQ ID NO: 1), GENBANK Accession No.NT_010966.10 truncated from nucleotides 2009236 to 2017289 (incorporatedherein as SEQ ID NO: 2); exons 1-4 extracted from the rhesus monkeygenomic sequence GENBANK Accession No. NW_001105671.1, based onsimilarity to human exons; and GENBANK Accession No. NW_001105671.1truncated from nucleotides 628000 to 638000 (incorporated herein as SEQID NO: 4).

Certain embodiments provide compounds comprising a modifiedoligonucleotide consisting of 8 to 80 linked nucleosides wherein thelinked nucleosides comprise at least 8 contiguous nucleobases of asequence selected from among the nucleobase sequences recited in SEQ IDNOs: 25, 80, 86, 87, 115, 120, 122, and 124. In certain embodiments, themodified oligonucleotide comprises at least 9, at least 10, at least 11,at least 12, at least 13, at least 14, at least 15, at least 16, atleast 17, at least 18, at least 19, or at least 20 contiguousnucleobases of a sequence selected from among the nucleobase sequencesrecited in SEQ ID NOs: 25, 80, 86, 87, 115, 120, 122, and 124.

Certain embodiments provide compounds comprising a modifiedoligonucleotide consisting of 12 to 50 linked nucleosides wherein thelinked nucleosides comprise at least 8, at least 9, at least 10, atleast 11, at least 12, at least 13, at least 14, at least 15, at least16, at least 17, at least 18, at least 19, or at least 20 contiguousnucleobases of a sequence selected from among the nucleobase sequencesrecited in SEQ ID NOs: 25, 80, 86, 87, 115, 120, 122, and 124. Incertain embodiments, the modified oligonucleotide comprises at least 9,at least 10, at least 11, at least 12, at least 13, at least 14, atleast 15, at least 16, at least 17, at least 18, at least 19, or atleast 20 contiguous nucleobases of a sequence selected from among thenucleobase sequences recited in SEQ ID NOs: 25, 80, 86, 87, 115, 120,122, and 124.

Certain embodiments provide compounds comprising a modifiedoligonucleotide consisting of 12 to 30 linked nucleosides wherein thelinked nucleosides comprise at least 8 contiguous nucleobases of asequence selected from among the nucleobase sequences recited in SEQ IDNOs: 25, 80, 86, 87, 115, 120, 122, and 124. In certain embodiments, themodified oligonucleotide comprises at least 9, at least 10, at least 11,at least 12, at least 13, at least 14, at least 15, at least 16, atleast 17, at least 18, at least 19, or at least 20 contiguousnucleobases of a sequence selected from among the nucleobase sequencesrecited in SEQ ID NOs: 25, 80, 86, 87, 115, 120, 122, and 124.

Certain embodiments provide compounds comprising a modifiedoligonucleotide consisting of 15 to 25 linked nucleosides wherein thelinked nucleosides comprise at least 8 contiguous nucleobases of asequence selected from among the nucleobase sequences recited in SEQ IDNOs: 25, 80, 86, 87, 115, 120, 122, and 124.

Certain embodiments provide compounds comprising a modifiedoligonucleotide consisting of 18 to 21 linked nucleosides wherein thelinked nucleosides comprise at least 8 contiguous nucleobases of asequence selected from among the nucleobase sequences recited in SEQ IDNOs: 25, 80, 86, 87, 115, 120, 122, and 124. In certain embodiments, themodified oligonucleotide comprises at least 9, at least 10, at least 11,at least 12, at least 13, at least 14, at least 15, at least 16, atleast 17, at least 18, at least 19, or at least 20 contiguousnucleobases of a sequence selected from among the nucleobase sequencesrecited in SEQ ID NOs: 25, 80, 86, 87, 115, 120, 122, and 124.

Certain embodiments provide compounds comprising a modifiedoligonucleotide consisting of 8 to 80 linked nucleosides wherein thelinked nucleosides comprise at least 8 contiguous nucleobases of asequence selected from among the nucleobase sequences recited in SEQ IDNOs: 25, 80, 86, and 87. In certain embodiments, the modifiedoligonucleotide comprises at least 9, at least 10, at least 11, at least12, at least 13, at least 14, at least 15, at least 16, at least 17, atleast 18, at least 19, or at least 20 contiguous nucleobases of asequence selected from among the nucleobase sequences recited in SEQ IDNOs: 25, 80, 86, and 87.

Certain embodiments provide compounds comprising a modifiedoligonucleotide consisting of 12 to 50 linked nucleosides wherein thelinked nucleosides comprise at least 8 contiguous nucleobases of asequence selected from among the nucleobase sequences recited in SEQ IDNOs: 25, 80, 86, and 87. In certain embodiments, the modifiedoligonucleotide comprises at least 9, at least 10, at least 11, at least12, at least 13, at least 14, at least 15, at least 16, at least 17, atleast 18, at least 19, or at least 20 contiguous nucleobases of asequence selected from among the nucleobase sequences recited in SEQ IDNOs: 25, 80, 86, and 87.

Certain embodiments provide compounds comprising a modifiedoligonucleotide consisting of 12 to 30 linked nucleosides wherein thelinked nucleosides comprise at least 8 contiguous nucleobases of asequence selected from among the nucleobase sequences recited in SEQ IDNOs: 25, 80, 86, and 87. In certain embodiments, the modifiedoligonucleotide comprises at least 9, at least 10, at least 11, at least12, at least 13, at least 14, at least 15, at least 16, at least 17, atleast 18, at least 19, or at least 20 contiguous nucleobases of asequence selected from among the nucleobase sequences recited in SEQ IDNOs: 25, 80, 86, and 87.

Certain embodiments provide compounds comprising a modifiedoligonucleotide consisting of 15 to 25 linked nucleosides wherein thelinked nucleosides comprise at least 8 contiguous nucleobases of asequence selected from among the nucleobase sequences recited in SEQ IDNOs: 25, 80, 86, and 87. In certain embodiments, the modifiedoligonucleotide comprises at least 9, at least 10, at least 11, at least12, at least 13, at least 14, at least 15, at least 16, at least 17, atleast 18, at least 19, or at least 20 contiguous nucleobases of asequence selected from among the nucleobase sequences recited in SEQ IDNOs: 25, 80, 86, and 87.

Certain embodiments provide compounds comprising a modifiedoligonucleotide consisting of 18 to 21 linked nucleosides wherein thelinked nucleosides comprise at least 8 contiguous nucleobases of asequence selected from among the nucleobase sequences recited in SEQ IDNOs: 25, 80, 86, and 87. In certain embodiments, the modifiedoligonucleotide comprises at least 9, at least 10, at least 11, at least12, at least 13, at least 14, at least 15, at least 16, at least 17, atleast 18, at least 19 or at least 20 contiguous nucleobases of asequence selected from among the nucleobase sequences recited in SEQ IDNOs: 25, 80, 86, and 87.

Certain embodiments provide compounds comprising a modifiedoligonucleotide consisting of 8 to 80 linked nucleosides wherein thelinked nucleosides comprise at least 8 contiguous nucleobases of thenucleobase sequence recited in SEQ ID NO: 80. In certain embodiments,the modified oligonucleotide comprises at least 9, at least 10, at least11, at least 12, at least 13, at least 14, at least 15, at least 16, atleast 17, at least 18, at least 19, or at least 20 contiguousnucleobases of a sequence selected from among the nucleobase sequencesrecited in SEQ ID NOs: 80.

Certain embodiments provide compounds comprising a modifiedoligonucleotide consisting of 12 to 50 linked nucleosides wherein thelinked nucleosides comprise at least 8 contiguous nucleobases of thenucleobase sequence recited in SEQ ID NO: 80. In certain embodiments,the modified oligonucleotide comprises at least 9, at least 10, at least11, at least 12, at least 13, at least 14, at least 15, at least 16, atleast 17, at least 18, at least 19, or at least 20 contiguousnucleobases of a sequence selected from among the nucleobase sequencesrecited in SEQ ID NOs: 80.

Certain embodiments provide compounds comprising a modifiedoligonucleotide consisting of 12 to 30 linked nucleosides wherein thelinked nucleosides comprise at least 8 contiguous nucleobases of thenucleobase sequence recited in SEQ ID NO: 80. In certain embodiments,the modified oligonucleotide comprises at least 9, at least 10, at least11, at least 12, at least 13, at least 14, at least 15, at least 16, atleast 17, at least 18, at least 19, or at least 20 contiguousnucleobases of a sequence selected from among the nucleobase sequencesrecited in SEQ ID NOs: 80.

Certain embodiments provide compounds comprising a modifiedoligonucleotide consisting of 15 to 25 linked nucleosides wherein thelinked nucleosides comprise at least 8 contiguous nucleobases of thenucleobase sequence recited in SEQ ID NO: 80. In certain embodiments,the modified oligonucleotide comprises at least 9, at least 10, at least11, at least 12, at least 13, at least 14, at least 15, at least 16, atleast 17, at least 18, at least 19, or at least 20 contiguousnucleobases of a sequence selected from among the nucleobase sequencesrecited in SEQ ID NOs: 80.

Certain embodiments provide compounds comprising a modifiedoligonucleotide consisting of 18 to 21 linked nucleosides wherein thelinked nucleosides comprise at least 8 contiguous nucleobases of thenucleobase sequence recited in SEQ ID NO: 80. In certain embodiments,the modified oligonucleotide comprises at least 9, at least 10, at least11, at least 12, at least 13, at least 14, at least 15, at least 16, atleast 17, at least 18, at least 19, or at least 20 contiguousnucleobases of a sequence selected from among the nucleobase sequencesrecited in SEQ ID NOs: 80.

Certain embodiments provide compounds comprising a modifiedoligonucleotide consisting of 20 to 30 linked nucleosides wherein thelinked nucleosides comprise at least 8 contiguous nucleobases of thenucleobase sequence recited in SEQ ID NO: 80. In certain embodiments,the modified oligonucleotide comprises at least 9, at least 10, at least11, at least 12, at least 13, at least 14, at least 15, at least 16, atleast 17, at least 18, or at least 19 contiguous nucleobases of thenucleobase sequence recited in SEQ ID NO: 80.

In certain embodiments, the compound comprises a modifiedoligonucleotide consisting of 20 linked nucleosides recited in SEQ IDNO: 80.

Certain embodiments provide compounds comprising a modifiedoligonucleotide consisting of 12 to 30 linked nucleosides wherein thelinked nucleosides comprise at least an 8 contiguous nucleobase portionthat is complementary to an equal length nucleobase portion within theregion selected from nucleotides 120-139, 212-236, 226-245, 293-468,293-326, 347-381, 425-468, 425-467, 452-478, 452-474, 459-478, 461-519,462-500, 500-519, 501-535, 502-531, 505-524, 507-526, 508-527, 514-540,514-539, 515-534, 516-535, 523-542, 544-606, 544-564, 564-583, 578-601,580-608, 580-599, 584-606, 585-604, 587-606, or 597-617 of SEQ ID NO: 1.In certain embodiments the region is selected from 507-526, 508-527,515-534, 516-535, 580-599, 585-604, 587-606 and 589-608 of SEQ ID NO: 1.In certain embodiments the region is selected from 501-535 or 580-608 ofSEQ ID NO: 1. In certain embodiments, the modified oligonucleotide hasat least a 9, at least a 10, at least an 11, at least an 12, at least an13, at least an 14, at least an 15, at least an 16, at least an 17, atleast an 18, at least an 19 or at least a 20 contiguous nucleobaseportion of which is complementary within a region described herein.

Certain embodiments provide compounds comprising a modifiedoligonucleotide consisting of 12 to 30 linked nucleosides wherein thelinked nucleosides comprise at least an 8 contiguous nucleobase portionthat is complementary to an equal length nucleobase portion within theregion selected from nucleotides 501-535 or 580-608 of SEQ ID NO: 1. Incertain embodiments, the modified oligonucleotide has at least a 9, atleast a 10, at least an 11, at least an 12, at least an 13, at least an14, at least an 15, at least an 16, at least an 17, at least an 18, atleast an 19 or at least a 20 contiguous nucleobase portion of which iscomplementary within a region described herein.

Certain embodiments provide compounds comprising a modifiedoligonucleotide consisting of 12 to 30 linked nucleosides wherein thelinked nucleosides comprise at least an 8 contiguous nucleobase portionthat is complementary to an equal length nucleobase portion within theregion selected from nucleotides 508-527 of SEQ ID NO: 1. In certainembodiments, the modified oligonucleotide has at least a 9, at least a10, at least an 11, at least an 12, at least an 13, at least an 14, atleast an 15, at least an 16, at least an 17, at least an 18, at least an19 or at least a 20 contiguous nucleobase portion of which iscomplementary within a region described herein.

Certain embodiments provide compounds comprising a modifiedoligonucleotide consisting of 15 to 25 linked nucleosides wherein thelinked nucleosides comprise at least an 8 contiguous nucleobase portionthat is complementary to an equal length nucleobase portion within theregion selected from nucleotides 507-526, 508-527, 515-534, 516-535,580-599, 585-604, 587-606 and 589-608 of SEQ ID NO: 1. In certainembodiments, the modified oligonucleotide has at least a 9, at least a10, at least an 11, at least an 12, at least an 13, at least an 14, atleast an 15, at least an 16, at least an 17, at least an 18, at least an19 or at least a 20 contiguous nucleobase portion of which iscomplementary within a region described herein.

Certain embodiments provide compounds comprising a modifiedoligonucleotide consisting of 18 to 21 linked nucleosides wherein thelinked nucleosides comprise at least an 8 contiguous nucleobase portionthat is complementary to an equal length nucleobase portion within theregion selected from nucleotides 507-526, 508-527, 515-534, 516-535,580-599, 585-604, 587-606 and 589-608 of SEQ ID NO: 1. In certainembodiments, the modified oligonucleotide has at least a 9, at least a10, at least an 11, at least an 12, at least an 13, at least an 14, atleast an 15, at least an 16, at least an 17, at least an 18, at least an19 or at least a 20 contiguous nucleobase portion of which iscomplementary within a region described herein.

Certain embodiments provide compounds comprising a modifiedoligonucleotide consisting of 20 linked nucleosides wherein the linkednucleosides comprise at least an 8 contiguous nucleobase portion that iscomplementary to an equal length nucleobase portion within the regionselected from nucleotides 507-526, 508-527, 515-534, 516-535, 580-599,585-604, 587-606 and 589-608 of SEQ ID NO: 1. In certain embodiments,the modified oligonucleotide has at least a 9, at least a 10, at leastan 11, at least a 12, at least a 13, at least a 14, at least a 15, atleast a 16, at least a 17, at least an 18, at least 19 or at least a 20contiguous nucleobase portion of which is complementary within a regiondescribed herein. In certain embodiments, the modified oligonucleotideis 90%, 95%, 99%, or 100% complementary to a nucleic acid encoding humantransthyretin (TTR), eg. SEQ ID No: 1.

Certain embodiments provide compounds comprising a modifiedoligonucleotide consisting of 20 linked nucleosides wherein the linkednucleosides comprise at least an 8 contiguous nucleobase portion that iscomplementary to an equal length nucleobase portion within the regionselected from nucleotides 508-527 of SEQ ID NO: 1. In certainembodiments, the modified oligonucleotide has at least a 9, at least a10, at least an 11, at least a 12, at least a 13, at least a 14, atleast a 15, at least a 16, at least a 17, at least an 18, at least 19 orat least a 20 contiguous nucleobase portion of which is complementary toan equal length portion within the region selected from nucleotides508-527 of SEQ ID NO: 1. In certain embodiments, the modifiedoligonucleotide is 90%, 95%, 99%, or 100% complementary to a nucleicacid encoding human transthyretin (TTR), eg. SEQ ID No: 1

Certain embodiments provide compounds comprising a modifiedoligonucleotide consisting of 20 linked nucleosides 60% complementarywithin the region selected from nucleotides 507-526, 508-527, 515-534,516-535, 580-599, 585-604, 587-606 and 589-608 of SEQ ID NO: 1.

Certain embodiments provide compounds comprising a modifiedoligonucleotide consisting of 20 linked nucleosides 70% complementarywithin the region selected from nucleotides 507-526, 508-527, 515-534,516-535, 580-599, 585-604, 587-606 and 589-608 of SEQ ID NO: 1.

Certain embodiments provide compounds comprising a modifiedoligonucleotide consisting of 20 linked nucleosides 80% complementarywithin the region selected from nucleotides 507-526, 508-527, 515-534,516-535, 580-599, 585-604, 587-606 and 589-608 of SEQ ID NO: 1.

Certain embodiments provide compounds comprising a modifiedoligonucleotide consisting of 20 linked nucleosides 90% complementarywithin the region selected from nucleotides 507-526, 508-527, 515-534,516-535, 580-599, 585-604, 587-606 and 589-608 of SEQ ID NO: 1.

Certain embodiments provide compounds comprising a modifiedoligonucleotide consisting of 20 linked nucleosides 95% complementarywithin the region selected from nucleotides 507-526, 508-527, 515-534,516-535, 580-599, 585-604, 587-606 and 589-608 of SEQ ID NO: 1.

Certain embodiments provide compounds comprising a modifiedoligonucleotide consisting of 20 linked nucleosides 99% complementarywithin the region selected from nucleotides 507-526, 508-527, 515-534,516-535, 580-599, 585-604, 587-606 and 589-608 of SEQ ID NO: 1.

Certain embodiments provide compounds comprising a modifiedoligonucleotide consisting of 20 linked nucleosides 100% complementarywithin the region selected from nucleotides 507-526, 508-527, 515-534,516-535, 580-599, 585-604, 587-606 and 589-608 of SEQ ID NO: 1.

Certain embodiments provide compounds comprising a modifiedoligonucleotide consisting of 20 linked nucleosides 60% complementarywithin nucleotides 508-527 of SEQ ID NO: 1.

Certain embodiments provide compounds comprising a modifiedoligonucleotide consisting of 20 linked nucleosides 70% complementarywithin nucleotides 508-527 of SEQ ID NO: 1.

Certain embodiments provide compounds comprising a modifiedoligonucleotide consisting of 20 linked nucleosides 80% complementarywithin nucleotides 508-527 of SEQ ID NO: 1.

Certain embodiments provide compounds comprising a modifiedoligonucleotide consisting of 20 linked nucleosides 90% complementarywithin nucleotides 508-527 of SEQ ID NO: 1.

Certain embodiments provide compounds comprising a modifiedoligonucleotide consisting of 20 linked nucleosides 95% complementarywithin nucleotides 508-527 of SEQ ID NO: 1.

Certain embodiments provide compounds comprising a modifiedoligonucleotide consisting of 20 linked nucleosides 99% complementarywithin nucleotides 508-527 of SEQ ID NO: 1.

Certain embodiments provide compounds comprising a modifiedoligonucleotide consisting of 20 linked nucleosides 100% complementarywithin nucleotides 508-527 of SEQ ID NO: 1.

In certain embodiments, an antisense compound or modifiedoligonucleotide targeted to a transthyretin nucleic acid targets thefollowing nucleotide regions of SEQ ID NO: 1: 120-139, 212-236, 226-245,293-468, 293-326, 347-381, 425-468, 425-467, 452-478, 452-474, 459-478,461-519, 462-500, 500-519, 502-531, 507-526, 505-524, 508-527, 514-540,514-539, 515-534, 516-535, 523-542, 544-606, 544-564, 564-583, 578-601,580-599, 584-606, 585-604, 587-606, or 597-617.

In certain embodiments, antisense compounds or modified oligonucleotidestargets a region of a transthyretin nucleic acid. In certainembodiments, such compounds or oligonucleotides targeted to a region ofa transthyretin nucleic acid have a contiguous nucleobase portion thatis complementary to an equal length nucleobase portion of the region.For example, the portion can be at least an 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19 or 20 contiguous nucleobases portion complementary toan equal length portion of a region recited herein. In certainembodiments, such compounds or oligonucleotide target the followingnucleotide regions of SEQ ID NO: 1: 120-139, 212-236, 226-245, 293-381,293-366, 353-381, 293-468, 425-468, 425-467, 452-476, 461-481, 461-500,500-519, 461-519, 502-531, 502-539, 504-536, 505-525, 506-530, 507-527,508-527, 508-536, 514-540, 523-542, 544-606, 544-564, 544-583, or597-617.

In certain embodiments, such compounds or oligonucleotides targeted to aregion of a transthyretin nucleic acid have a contiguous nucleobaseportion that is complementary to an equal length nucleobase portion ofthe region 501-535 or 580-608 of SEQ ID NO: 1.

In certain embodiments, the following nucleotide regions of SEQ ID NO:1, when targeted by antisense compounds or oligonucleotides, displays atleast 60% inhibition: 226-245, 293-366, 357-467, 452-474, 457-476,459-478, 462-500, 500-519, 502-531, 504-536, 505-525, 506-530, 507-527,508-527, 508-536, 514-539, 544-564, 564-583, 578-601, 584-606, or597-617.

In certain embodiments, the following nucleotide regions of SEQ ID NO:1, when targeted by antisense compounds or oligonucleotides, displays atleast 65% inhibition: 293-366, 357-376, 425-449, 432-467, 452-474,459-478, 462-500, 500-519, 502-531, 504-536, 505-525, 506-530, 507-527,508-527, 508-536, 514-539, 544-563, 564-583, 578-601,585-606, or597-617.

In certain embodiments, the following nucleotide regions of SEQ ID NO:1, when targeted by antisense compounds or oligonucleotides, displays atleast 70% inhibition: 293-366, 425-449, 432-467, 452-474, 459-478,462-500, 500-519, 502-531, 504-536, 505-525, 506-530, 507-527, 508-527,508-536, 514-539, 564-583, 578-598, 581-600, or 597-617.

In certain embodiments, the following nucleotide regions of SEQ ID NO:1, when targeted by antisense compounds or oligonucleotides, displays atleast 75% inhibition: 293-322, 347-366, 425-449, 432-467, 452-474,459-478, 462-500, 500-519, 503-531, 504-536, 505-525, 506-530, 507-527,508-527, 508-536, 514-539, 578-598, 581-600, or 597-616.

In certain embodiments, the following nucleotide regions of SEQ ID NO:1, when targeted by antisense compounds or oligonucleotides, displays atleast 80% inhibition: 303-322, 425-449, 432-460, 443-467, 452-473,481-500, 500-519, 503-531, 504-536, 505-525, 506-530, 507-527, 508-527,508-536, 514-536, 519-539, 579-598, 581-600, or 597-616.

In certain embodiments, the following nucleotide regions of SEQ ID NO:1, when targeted by antisense compounds or oligonucleotides, displays atleast 85% inhibition: 427-449, 432-458, 441-460, 443-467, 452-473,504-531, 504-536, 505-525, 506-530, 507-527, 508-527, 508-536, 514-536,519-539, or 581-600.

In certain embodiments, the following nucleotide regions of SEQ ID NO:1, when targeted by antisense compounds or oligonucleotides, displays atleast 90% inhibition: 428-449, 432-456, 439-458, 441-460, 445-466,452-473, 504-525, 508-527, or 515-536.

In certain embodiments, the following nucleotide regions of SEQ ID NO:1, when targeted by antisense compounds or oligonucleotides, displays atleast 95% inhibition: 434-453, 436-456, 441-460, 445-465, 505-524, or516-535.

In certain embodiments, the following antisense compounds target aregion of a SEQ ID NO: 1, a nucleic acid encoding human transthyretin,and demonstrate at least 60% inhibition of a transthyretin mRNA: ISISNOs:.420954, 420904, 304286, 420874, 420948, 420883, 420955, 420952,420956, 420957, 420882, 420947, 420950, 304312, 304307, 420879, 420910,420902, 420908, 420924, 420877, 420880, 304309, 304289, 420906, 304311,420878, 420911, 304284, 304288, 420909, 304296, 420949, 304290, 304299,420898, 420920, 420925, 420951, 304287, 420894, 420916, 420918, 420926,304285, 420919, 420923, 420886, 420900, 420912, 420915, 420917, 420921,420884, 420885, 420887, 420889, 420892, 420901, 420914, 420897, 420899,420888, 420895, 420896, 420913, 420922, 420893, 420890, or 420891.

In certain embodiments, the following antisense compounds target aregion of a SEQ ID NO: 1, a nucleic acid encoding human transthyretinand demonstrate at least 65% inhibition of a transthyretin mRNA: ISISNOs: 420955, 420952, 420956, 420957, 420882, 420947, 420950, 304312,304307, 420879, 420910, 420902, 420908, 420924, 420877, 420880, 304309,304289, 420906, 304311, 420878, 420911, 304284, 304288, 420909, 304296,420949, 304290, 304299, 420898, 420920, 420925, 420951, 304287, 420894,420916, 420918, 420926, 304285, 420919, 420923, 420886, 420900, 420912,420915, 420917, 420921, 420884, 420885, 420887, 420889, 420892, 420901,420914, 420897, 420899, 420888, 420895, 420896, 420913, 420922, 420893,420890, or 420891.

In certain embodiments, the following antisense compounds target aregion of a SEQ ID NO: 1, a nucleic acid encoding human transthyretinand demonstrate at least 70% inhibition of a transthyretin mRNA: ISISNOs: 304312, 304307, 420879, 420910, 420902, 420908, 420924, 420877,420880, 304309, 304289, 420906, 304311, 420878, 420911, 304284, 304288,420909, 304296, 420949, 304290, 304299, 420898, 420920, 420925, 420951,304287, 420894, 420916, 420918, 420926, 304285, 420919, 420923, 420886,420900, 420912, 420915, 420917, 420921, 420884, 420885, 420887, 420889,420892, 420901, 420914, 420897, 420899, 420888, 420895, 420896, 420913,420922, 420893, 420890, or 420891.

In certain embodiments, the following antisense compounds target aregion of a SEQ ID NO: 1, a nucleic acid encoding human transthyretinand demonstrate at least 75% inhibition of a transthyretin mRNA: ISISNOs: 420877, 420878, 420880, 304284, 304285, 420884, 420885, 420886,420887, 420888, 420889, 420890, 420891, 304287, 420892, 304288, 420893,304289, 304290, 420894, 420895, 420896, 420897, 420898, 420899, 420900,420901, 420902, 420906, 420908, 304296, 420909, 420911, 420912, 420913,420914, 304299, 420915, 420916, 420917, 420918, 420919, 420920, 420921,420922, 420923, 420924, 420925, 420926, 304309, 420949, 420951, or304311.

In certain embodiments, the following antisense compounds target aregion of a SEQ ID NO: 1, a nucleic acid encoding human transthyretinand demonstrate at least 80% inhibition of a transthyretin mRNA: ISISNOs: 304311, 420878, 420911, 304284, 304288, 420909, 304296, 420949,304290, 304299, 420898, 420920, 420925, 420951, 304287, 420894, 420916,420918, 420926, 304285, 420919, 420923, 420886, 420900, 420912, 420915,420917, 420921, 420884, 420885, 420887, 420889, 420892, 420901, 420914,420897, 420899, 420888, 420895, 420896, 420913, 420922, 420893, 420890,or 42089.

In certain embodiments, the following antisense compounds target aregion of a SEQ ID NO: 1, a nucleic acid encoding human transthyretinand demonstrate at least 85% inhibition of a transthyretin mRNA: ISISNOs: 304290, 304299, 420898, 420920, 420925, 420951, 304287, 420894,420916, 420918, 420926, 304285, 420919, 420923, 420886, 420900, 420912,420915, 420917, 420921, 420884, 420885, 420887, 420889, 420892, 420901,420914, 420897, 420899, 420888, 420895, 420896, 420913, 420922, 420893,420890, or 420891.

In certain embodiments, the following antisense compounds target aregion of a SEQ ID NO: 1, a nucleic acid encoding human transthyretinand demonstrate at least 90% inhibition of a transthyretin mRNA: ISISNOs: 420923, 420886, 420900, 420912, 420915, 420917, 420921, 420884,420885, 420887, 420889, 420892, 420901, 420914, 420897, 420899, 420888,420895, 420896, 420913, 420922, 420893, 420890, or 420891.

In certain embodiments, the following antisense compounds target aregion of a SEQ ID NO: 1, a nucleic acid encoding human transthyretinand demonstrate at least 95% inhibition of a transthyretin mRNA: ISISNOs: 420888, 420895, 420896, 420913, 420922, 420893, 420890, or 420891.

In certain embodiments, a target region is nucleotides 120-139 of SEQ IDNO: 1. In certain embodiments, an antisense compound is targeted tonucleotides 120-139 of SEQ ID NO: 1. In certain embodiments, anantisense compound targeted to a transthyretin nucleic acid comprises anucleobase sequence selected from SEQ ID NO: 37. In certain suchembodiments, an antisense compound targeted to nucleotides 120-139 ofSEQ ID NO: 1 is selected from ISIS NO: 420872.

In certain embodiments, a target region is nucleotides 212-236 of SEQ IDNO: 1. In certain embodiments, an antisense compound is targeted tonucleotides 212-236 of SEQ ID NO: 1. In certain embodiments, anantisense compound targeted to a transthyretin nucleic acid comprises anucleobase sequence selected from SEQ ID NOs: 8 and 38. In certain suchembodiments, an antisense compound targeted to nucleotides 212-236 ofSEQ ID NO: 1 is selected from ISIS NOs: 420873 or 304267.

In certain embodiments, a target region is nucleotides 226-245 of SEQ IDNO: 1. In certain embodiments, an antisense compound is targeted tonucleotides 226-245 of SEQ ID NO: 1. In certain embodiments, anantisense compound targeted to a transthyretin nucleic acid comprises anucleobase sequence selected from SEQ ID NO: 39. In certain suchembodiments, an antisense compound targeted to nucleotides 226-245 ofSEQ ID NO: 1 is selected from ISIS NO: 420874.

In certain embodiments, a target region is nucleotides 293-381 of SEQ IDNO: 1. In certain embodiments, an antisense compound is targeted tonucleotides 293-381 of SEQ ID NO: 1. In certain embodiments, anantisense compound targeted to a transthyretin nucleic acid comprises anucleobase sequence selected from SEQ ID NOs: 10, 42-48. In certain suchembodiments, an antisense compound targeted to nucleotides 293-381 ofSEQ ID NO: 1 is selected from ISIS NOs: 420877, 420878, 420879, 420880,304280, 420881, 420882, or 420883.

In certain embodiments, a target region is nucleotides 293-366 of SEQ IDNO: 1. In certain embodiments, an antisense compound is targeted tonucleotides 293-366 of SEQ ID NO: 1. In certain embodiments, anantisense compound targeted to a transthyretin nucleic acid comprises anucleobase sequence selected from SEQ ID NOs: 42-45. In certain suchembodiments, an antisense compound targeted to nucleotides 293-366 ofSEQ ID NO: 1 is selected from ISIS NOs: 420877, 420878, 420879, or420880.

In certain embodiments, a target region is nucleotides 353-381 of SEQ IDNO: 1. In certain embodiments, an antisense compound is targeted tonucleotides 353-381 of SEQ ID NO: 1. In certain embodiments, anantisense compound targeted to a transthyretin nucleic acid comprises anucleobase sequence selected from SEQ ID NOs: 10, 46-48. In certain suchembodiments, an antisense compound targeted to nucleotides 353-381 ofSEQ ID NO: 1 is selected from ISIS NOs: 304280, 420881, 420882, or420883.

In certain embodiments, a target region is nucleotides 293-468 of SEQ IDNO: 1. In certain embodiments, an antisense compound is targeted tonucleotides 293-468 of SEQ ID NO: 1. In certain embodiments, anantisense compound targeted to a transthyretin nucleic acid comprises anucleobase sequence selected from SEQ ID NOs: 10-18, 42-63. In certainsuch embodiments, an antisense compound targeted to nucleotides 293-468of SEQ ID NO: 1 is selected from ISIS NOs: 420877, 420878, 420879,420880, 304280, 420881, 420882, 420883, 304284, 304285, 420884, 420885,304286, 420886, 420887, 420888, 420889, 420890, 420891, 304287, 420892,304288, 420893, 304289, 304290, 420894, 420895, 420896, 420897, 420898,or 304291.

In certain embodiments, a target region is nucleotides 425-468 of SEQ IDNO: 1. In certain embodiments, an antisense compound is targeted tonucleotides 425-468 of SEQ ID NO: 1. In certain embodiments, anantisense compound targeted to a transthyretin nucleic acid comprises anucleobase sequence selected from SEQ ID NOs: 11-18, 49-63. In certainsuch embodiments, an antisense compound targeted to nucleotides 425-468of SEQ ID NO: 1 is selected from ISIS NOs: 304284, 304285, 420884,420885, 304286, 420886, 420887, 420888, 420889, 420890, 420891, 304287,420892, 304288, 420893, 304289, 304290, 420894, 420895, 420896, 420897,420898, or 304291.

In certain embodiments, a target region is nucleotides 425-467 of SEQ IDNO: 1. In certain embodiments, an antisense compound is targeted tonucleotides 425-468 of SEQ ID NO: 1. In certain embodiments, anantisense compound targeted to a transthyretin nucleic acid comprises anucleobase sequence selected from SEQ ID NOs: 11-17, 49-63. In certainsuch embodiments, an antisense compound targeted to nucleotides 425-468of SEQ ID NO: 1 is selected from ISIS NOs: 304284, 304285, 420884,420885, 304286, 420886, 420887, 420888, 420889, 420890, 420891, 304287,420892, 304288, 420893, 304289, 304290, 420894, 420895, 420896, 420897,or 420898.

In certain embodiments, a target region is nucleotides 452-476 of SEQ IDNO: 1. In certain embodiments, an antisense compound is targeted tonucleotides 452-476 of SEQ ID NO: 1. In certain embodiments, anantisense compound targeted to a transthyretin nucleic acid comprises anucleobase sequence selected from SEQ ID NOs: 64-69. In certain suchembodiments, an antisense compound targeted to nucleotides 452-476 ofSEQ ID NO: 1 is selected from ISIS NOs: 420889, 420890, 420891, 304287,420892, 304288, 420893, 304289, 304290, 420894, 420895, 420896, 420897,420898, 304291, 304292, 304293, 420899, 420900, 420901, 420902, 420903,or 420904.

In certain embodiments, a target region is nucleotides 461-481 of SEQ IDNO: 1. In certain embodiments, an antisense compound is targeted tonucleotides 461-481 of SEQ ID NO: 1. In certain embodiments, anantisense compound targeted to a transthyretin nucleic acid comprises anucleobase sequence selected from SEQ ID NOs: 72-73. In certain suchembodiments, an antisense compound targeted to nucleotides 461-481 ofSEQ ID NO: 1 is selected from ISIS NOs: 420907 or 420908.

In certain embodiments, a target region is nucleotides 461-500 of SEQ IDNO: 1. In certain embodiments, an antisense compound is targeted tonucleotides 461-500 of SEQ ID NO: 1. In certain embodiments, anantisense compound targeted to a transthyretin nucleic acid comprises anucleobase sequence selected from SEQ ID NOs: 22, 72 and 73. In certainsuch embodiments, an antisense compound targeted to nucleotides 461-500of SEQ ID NO: 1 is selected from ISIS NOs: 420907, 420908 or 304296.

In certain embodiments, a target region is nucleotides 500-519 of SEQ IDNO: 1. In certain embodiments, an antisense compound is targeted tonucleotides 500-519 of SEQ ID NO: 1. In certain embodiments, anantisense compound targeted to a transthyretin nucleic acid comprises anucleobase sequence selected from SEQ ID NO: 74. In certain suchembodiments, an antisense compound targeted to nucleotides 500-519 ofSEQ ID NO: 1 is selected from ISIS NO: 420909.

In certain embodiments, a target region is nucleotides 461-519 of SEQ IDNO: 1. In certain embodiments, an antisense compound is targeted tonucleotides 461-519 of SEQ ID NO: 1. In certain embodiments, anantisense compound targeted to a transthyretin nucleic acid comprises anucleobase sequence selected from SEQ ID NOs: 22, 23, 72-74. In certainsuch embodiments, an antisense compound targeted to nucleotides 461-519of SEQ ID NO: 1 is selected from ISIS NOs: 420907, 420908, 304296, or420909.

In certain embodiments, a target region is nucleotides 502-531 of SEQ IDNO: 1. In certain embodiments, an antisense compound is targeted tonucleotides 502-531 of SEQ ID NO: 1. In certain embodiments, anantisense compound targeted to a transthyretin nucleic acid comprises anucleobase sequence selected from SEQ ID NOs: 25, 75-84. In certain suchembodiments, an antisense compound targeted to nucleotides 502-531 ofSEQ ID NO: 1 is selected from ISIS NOs: 420910, 420911, 420912, 420913,420914, 304299, 420915, 420916, 420917, 420918, or 420919.

In certain embodiments, a target region is nucleotides 502-539 of SEQ IDNO: 1. In certain embodiments, an antisense compound is targeted tonucleotides 502-539 of SEQ ID NO: 1. In certain embodiments, anantisense compound targeted to a transthyretin nucleic acid comprises anucleobase sequence selected from SEQ ID NOs: 25, 26, 75-91. In certainsuch embodiments, an antisense compound targeted to nucleotides 502-539of SEQ ID NO: 1 is selected from ISIS NOs: 420910, 420911, 420912,420913, 420914, 304299, 420915, 420916, 420917, 420918, 420919, 304300,420920, 420921, 420922, 420923, 420924, 420925, or 420926.

In certain embodiments, a target region is nucleotides 504-536 of SEQ IDNO: 1. In certain embodiments, an antisense compound is targeted tonucleotides 504-536 of SEQ ID NO: 1. In certain embodiments, anantisense compound targeted to a transthyretin nucleic acid comprises anucleobase sequence selected from SEQ ID NOs: 25, 26, 77-88. In certainsuch embodiments, an antisense compound targeted to nucleotides 504-536of SEQ ID NO: 1 is selected from ISIS NOs: 420912, 420913, 420914,304299, 420915, 420916, 420917, 420918, 420919, 304300, 420920, 420921,420922, or 420923.

In certain embodiments, a target region is nucleotides 505-535 of SEQ IDNO: 1. In certain embodiments, an antisense compound is targeted tonucleotides 505-535 of SEQ ID NO: 1. In certain embodiments, anantisense compound targeted to a transthyretin nucleic acid comprises anucleobase sequence selected from SEQ ID NOs: 25, 26, 78-87. In certainsuch embodiments, an antisense compound targeted to nucleotides 505-535of SEQ ID NO: 1 is selected from ISIS NOs: 420913, 420914, 304299,420915, 420916, 420917, 420918, 420919, 304300, 420920, 420921, or420922.

In certain embodiments, a target region is nucleotides 506-530 of SEQ IDNO: 1. In certain embodiments, an antisense compound is targeted tonucleotides 506-530 of SEQ ID NO: 1. In certain embodiments, anantisense compound targeted to a transthyretin nucleic acid comprises anucleobase sequence selected from SEQ ID NOs: 25, 79-83. In certain suchembodiments, an antisense compound targeted to nucleotides 506-530 ofSEQ ID NO: 1 is selected from ISIS NOs: 420913, 420914, 304299, 420915,420916, 420917, 420918, or 420919.

In certain embodiments, a target region is nucleotides 507-527 of SEQ IDNO: 1. In certain embodiments, an antisense compound is targeted tonucleotides 507-527 of SEQ ID NO: 1. In certain embodiments, anantisense compound targeted to a transthyretin nucleic acid comprises anucleobase sequence selected from SEQ ID NOs: 25 or 80. In certain suchembodiments, an antisense compound targeted to nucleotides 507-527 ofSEQ ID NO: 1 is selected from ISIS NO: 304299 or 420915.

In certain embodiments, a target region is nucleotides 508-527 of SEQ IDNO: 1. In certain embodiments, an antisense compound is targeted tonucleotides 508-527 of SEQ ID NO: 1. In certain embodiments, anantisense compound targeted to a transthyretin nucleic acid comprises anucleobase sequence selected from SEQ ID NO: 80. In certain suchembodiments, an antisense compound targeted to nucleotides 508-527 ofSEQ ID NO: 1 is selected from ISIS NO: 420915.

In certain embodiments, a target region is nucleotides 514-540 of SEQ IDNO: 1. In certain embodiments, an antisense compound is targeted tonucleotides 514-540 of SEQ ID NO: 1. In certain embodiments, anantisense compound targeted to a transthyretin nucleic acid comprises anucleobase sequence selected from SEQ ID NOs: 85-92. In certain suchembodiments, an antisense compound targeted to nucleotides 514-540 ofSEQ ID NO: 1 is selected from ISIS NOs: 420920, 420921, 420922, 420923,420924, 420925, 420926, or 420927.

In certain embodiments, a target region is nucleotides 523-542 of SEQ IDNO: 1. In certain embodiments, an antisense compound is targeted tonucleotides 523-542 of SEQ ID NO: 1. In certain embodiments, anantisense compound targeted to a transthyretin nucleic acid comprises anucleobase sequence selected from SEQ ID NO: 94. In certain suchembodiments, an antisense compound targeted to nucleotides 523-542 ofSEQ ID NO: 1 is selected from ISIS NO: 420929.

In certain embodiments, a target region is nucleotides 544-606 of SEQ IDNO: 1. In certain embodiments, an antisense compound is targeted tonucleotides 544-606 of SEQ ID NO: 1. In certain embodiments, anantisense compound targeted to a transthyretin nucleic acid comprises anucleobase sequence selected from SEQ ID NOs: 30-33, 112-122. In certainsuch embodiments, an antisense compound targeted to nucleotides 544-606of SEQ ID NO: 1 is selected from ISIS NOs: 420947, 420948, 304304,304307, 304308, 304309, 420949, 420950, 420951, 420952, 420953, 420954,420955, 420956, or 420957.

In certain embodiments, a target region is nucleotides 544-564 of SEQ IDNO: 1. In certain embodiments, an antisense compound is targeted tonucleotides 544-564 of SEQ ID NO: 1. In certain embodiments, anantisense compound targeted to a transthyretin nucleic acid comprises anucleobase sequence selected from SEQ ID NOs: 112-113. In certain suchembodiments, an antisense compound targeted to nucleotides 544-564 ofSEQ ID NO: 1 is selected from ISIS NOs: 420947 or 420948.

In certain embodiments, a target region is nucleotides 544-583 of SEQ IDNO: 1. In certain embodiments, an antisense compound is targeted tonucleotides 544-583 of SEQ ID NO: 1. In certain embodiments, anantisense compound targeted to a transthyretin nucleic acid comprises anucleobase sequence selected from SEQ ID NOs: 30, 31, 112, and 113. Incertain such embodiments, an antisense compound targeted to nucleotides544-583 of SEQ ID NO: 1 is selected from ISIS NOs: 420947, 420948,304304, or 304307.

In certain embodiments, a target region is nucleotides 597-617 of SEQ IDNO: 1. In certain embodiments, an antisense compound is targeted tonucleotides 597-617 of SEQ ID NO: 1. In certain embodiments, anantisense compound targeted to a transthyretin nucleic acid comprises anucleobase sequence selected from SEQ ID NOs: 34-35. In certain suchembodiments, an antisense compound targeted to nucleotides 597-617 ofSEQ ID NO: 1 is selected from ISIS NOs: 304311 or 304312.

In certain embodiments, the modified oligonucleotide consists of asingle-stranded modified oligonucleotide.

In certain embodiments, the modified oligonucleotide consists of 20linked nucleosides.

In certain embodiments, the nucleobase sequence of the modifiedoligonucleotide is at least 90% complementary over its entire length toa nucleobase sequence of SEQ ID NO: 1, 2, or 4. In certain embodiments,the nucleobase sequence of the modified oligonucleotide is at least 95%complementary over its entire length to a nucleobase sequence of SEQ IDNO: 1, 2, or 4. In certain embodiments, the modified oligonucleotide isat least 99% complementary over its entire length to SEQ ID NO: 1, 2, or4. In certain embodiments, the nucleobase sequence of the modifiedoligonucleotide is 100% complementary over its entire length to anucleobase sequence of SEQ ID NO: 1, 2, or 4.

In certain embodiments, the compound has at least one modifiedinternucleoside linkage. In certain embodiments, the internucleosidelinkage is a phosphorothioate internucleoside linkage.

In certain embodiments, the compound has at least one nucleosidecomprising a modified sugar. In certain embodiments, the at least onemodified sugar is a bicyclic sugar. In certain embodiments, the at leastone bicyclic sugar comprises a 4′—CH(CH₃)—O—2′ bridge. In certainembodiments, the at least one modified sugar comprises a2′—O-methoxyethyl.

In certain embodiments, the compound comprises at least one at least onetetrahydropyran modified nucleoside wherein a tetrahydropyran ringreplaces the furanose ring. In certain embodiments, the at least onetetrahydropyran modified nucleoside has the structure:

wherein Bx is an optionally protected heterocyclic base moiety.

In certain embodiments, the compound has at least one nucleosidecomprising a modified nucleobase. In certain embodiments, the modifiednucleobase is a 5-methylcytosine.

In certain embodiments, the modified oligonucleotide of the compoundcomprises:

-   (i) a gap segment consisting of linked deoxynucleosides;-   (ii) a 5′ wing segment consisting of linked nucleosides;-   (iii) a 3′ wing segment consisting of linked nucleosides, wherein    the gap segment is positioned between the 5′ wing segment and the 3′    wing segment and wherein each nucleoside of each wing segment    comprises a modified sugar.

In certain embodiments, the modified oligonucleotide of the compoundcomprises:

-   (i) a gap segment consisting of ten linked deoxynucleosides;-   (ii) a 5′ wing segment consisting of five linked nucleosides;-   (iii) a 3′ wing segment consisting of five linked nucleosides,    wherein the gap segment is positioned immediately adjacent to and    between the 5′ wing segment and the 3′ wing segment, wherein each    nucleoside of each wing segment comprises a 2′—O-methoxyethyl sugar;    and wherein each internucleoside linkage is a phosphorothioate    linkage.

In certain embodiments, the modified oligonucleotide of the compoundcomprises:

-   (i) a gap segment consisting of eight linked deoxynucleosides;-   (ii) a 5′ wing segment consisting of six linked nucleosides;-   (iii) a 3′ wing segment consisting of six linked nucleosides,    wherein the gap segment is positioned immediately adjacent to and    between the 5′ wing segment and the 3′ wing segment, wherein each    nucleoside of each wing segment comprises a 2′—O-methoxyethyl sugar;    and wherein each internucleoside linkage is a phosphorothioate    linkage.

In certain embodiments, the modified oligonucleotide of the compoundcomprises:

-   (i) a gap segment consisting of eight linked deoxynucleosides;-   (ii) a 5′ wing segment consisting of five linked nucleosides;-   (iii) a 3′ wing segment consisting of five linked nucleosides,    wherein the gap segment is positioned immediately adjacent to and    between the 5′ wing segment and the 3′ wing segment, wherein each    nucleoside of each wing segment comprises a 2′—O-methoxyethyl sugar;    and wherein each internucleoside linkage is a phosphorothioate    linkage.

In certain embodiments, the modified oligonucleotide of the compoundcomprises:

-   (i) a gap segment consisting of ten linked deoxynucleosides;-   (ii) a 5′ wing segment consisting of five linked nucleosides;-   (iii) a 3′ wing segment consisting of five linked nucleosides,    wherein the gap segment is positioned immediately adjacent to and    between the 5′ wing segment and the 3′ wing segment, wherein each    nucleoside of each wing segment comprises a 2′—O-methoxyethyl sugar;    and wherein each internucleoside linkage is a phosphorothioate    linkage; and wherein the nucleobase sequence comprises at least 8    contiguous nucleobases of the nucleobase sequence recited in SEQ ID    NO: 80.

In certain embodiments, the modified oligonucleotide of the compoundcomprises:

-   (i) a gap segment consisting of ten linked deoxynucleosides;-   (ii) a 5′ wing segment consisting of five linked nucleosides;-   (iii) a 3′ wing segment consisting of five linked nucleosides,    wherein the gap segment is positioned immediately adjacent to and    between the 5′ wing segment and the 3′ wing segment, wherein each    nucleoside of each wing segment comprises a 2′—O-methoxyethyl sugar;    and wherein each internucleoside linkage is a phosphorothioate    linkage; and wherein the nucleobase sequence is recited in SEQ ID    NO: 80.

Certain embodiments provide a composition comprising a compound asdescribed herein, or a salt thereof, and a pharmaceutically acceptablecarrier or diluent. In certain embodiments, the composition comprises amodified oligonucleotide consisting of 12 to 30 linked nucleosides andhaving a nucleobase sequence comprising at least 12 contiguousnucleobases of a nucleobase sequence selected from among the nucleobasesequences recited in SEQ ID NOs: 25, 80, 86, 87, 115, 120, 122, and 124or a salt thereof and a pharmaceutically acceptable carrier or diluent.

Certain embodiments provide a composition comprising a compound asdescribed herein, or a salt thereof, and a pharmaceutically acceptablecarrier or diluent. In certain embodiments, the composition comprises amodified oligonucleotide consisting of 12 to 30 linked nucleosides andhaving a nucleobase sequence comprising at least 12 contiguousnucleobases of the nucleobase sequences recited in SEQ ID NO: 80 or asalt thereof and a pharmaceutically acceptable carrier or diluent.

Certain embodiments provide a composition comprising a compound asdescribed herein, wherein the viscosity level is less than 40 cP. Incertain embodiments, the composition has a viscosity level less than 15cP. In certain embodiments, the composition has a viscosity level lessthan 12 cP. In certain embodiments, the composition has a viscositylevel less than 10 cP.

Certain embodiments provide methods of treating, preventing, orameliorating transthyretin amyloidosis.

Certain embodiments provide methods comprising administering to ananimal a compound as described herein to an animal. In certainembodiments, the method comprises administering to an animal a modifiedoligonucleotide consisting of 12 to 30 linked nucleosides and having anucleobase sequence comprising at least 8 contiguous nucleobases of anucleobase sequence selected from among the nucleobase sequences recitedin SEQ ID NOs: 25, 80, 86, 87, 115, 120, 122, and 124.

Certain embodiments provide methods comprising administering to ananimal a compound as described herein to an animal. In certainembodiments, the method comprises administering to an animal a compoundor modified oligonucleotide consisting 12 to 30 linked nucleosides,wherein the linked nucleosides comprise at least an 8 contiguousnucleobase portion complementary to an equal length portion within theregion selected from nucleotides 501-535 or 580-608 of SEQ ID NO: 1.

Certain embodiments provide methods comprising administering to ananimal a compound as described herein to an animal. In certainembodiments, the method comprises administering to an animal a modifiedoligonucleotide consisting of 12 to 30 linked nucleosides and having anucleobase sequence comprising at least 8 contiguous nucleobases of anucleobase sequence recited in SEQ ID NO: 80.

Certain embodiments provide methods comprising administering to ananimal a compound as described herein to an animal. In certainembodiments, the method comprises administering to an animal a compoundor modified oligonucleotide consisting 12 to 30 linked nucleosides,wherein the linked nucleosides comprise at least an 8 contiguousnucleobase portion complementary to an equal length portion within theregion selected from nucleotides 508-527 of SEQ ID NO: 1.

In certain embodiments, the animal is a human.

In certain embodiments, the administering prevents, treats, ameliorates,or slows progression of transthyretin amyloidosis as described herein.

In certain embodiments, the compound is co-administered with a secondagent.

In certain embodiments, the compound and the second agent areadministered concomitantly.

In certain embodiments, the administering is parenteral administration.In certain embodiments, the parenteral administration is subcutaneousadministration. In certain embodiments, the formulation foradministering is the compound in saline. In certain embodiments, thecompound comprises a modified oligonucleotide consisting of 12 to 30linked nucleosides and having a nucleobase sequence comprising at least12 contiguous nucleobases of a nucleobase sequence selected from amongthe nucleobase sequences recited in SEQ ID NOs: 25, 80, 86, 87, 115,120, 122, and 124 or a salt thereof and saline. In certain embodiments,the formulation does not include any stabilizing agents or additionalstabilizing agents including lipid agents.

In certain embodiments, the administering is parenteral administration.In certain embodiments, the parenteral administration is intracranialadministration. In certain embodiments, the intracranial administrationis intracerebral, intrathecal, intraventricular, ventricular,intracerebroventricular, cerebral intraventricular or cerebralventricular administration.

Certain embodiments further provide a method to reduce transthyretinmRNA or protein expression in an animal comprising administering to theanimal a compound or composition as described herein to reducetransthyretin mRNA or protein expression in the animal. In certainembodiments, the animal is a human. In certain embodiments, reducingtransthyretin mRNA or protein expression prevents, treats, ameliorates,or slows progression of transthyretin amyloidosis.

Certain embodiments provide a method for treating a human with atransthyretin related disease comprising identifying the human with thedisease and administering to the human a therapeutically effectiveamount of a compound or composition as described herein. In certainembodiments, the treatment reduces a symptom selected from the groupconsisting of restlessness, lack of coordination, nystagmus, spasticparaparesis, lack of muscle coordination, impaired vision, insomnia,unusual sensations, myoclonus, blindness, loss of speech, Carpal tunnelsyndrome, seizures, subarachnoid hemorrhages, stroke and bleeding in thebrain, hydrocephalus, ataxia, and spastic paralysis, coma, sensoryneuropathy, parathesia, hypesthesia, motor neuropathy, autonomicneuropathy, orthostatic hypotension, cyclic constipation, cyclicdiarrhea, nausea, vomiting, reduced sweating, impotence, delayed gastricemptying, urinary retention, urinary incontinence, progressivecardiopathy, fatigue, shortness of breath, weight loss, lack ofappetite, numbness, tingling, weakness, enlarged tongue, nephroticsyndrome, congestive heart failure, dyspnea on exertion, peripheraledema, arrhythmias, palpitations, light-headedness, syncope, posturalhypotension, peripheral nerve problems, sensory motor impairment, lowerlimb neuropathy, upper limb neuropathy, hyperalgesia, alteredtemperature sensation, lower extremity weakness, cachexia, peripheraledema, hepatomegaly, purpura, diastolic dysfunction, prematureventricular contractions, cranial neuropathy, diminished deep tendonreflexes, amyloid deposits in the corpus vitreum, vitreous opacity, dryeyes, glaucoma, scalloped appearance in the pupils, swelling of the feetdue to water retention. In certain embodiments, the symptom is acognitive symptom selected from the group consisting of impaired memory,impaired judgment, and thinking, impaired planning, impairedflexibility, impaired abstract thinking, impaired rule acquisition,impaired initiation of appropriate actions, impaired inhibition ofinappropriate actions, impaired short-term memory, impaired long-termmemory, paranoia, disorientation, confusion, hallucination and dementia.In certain embodiments, the symptom is a psychiatric symptom selectedfrom the group consisting of dementia; anxiety, depression, bluntedaffect, egocentrisms, aggression, compulsive behavior, irritability,personality changes, including, impaired memory, judgment, and thinkingand suicidal ideation.

Further embodiments provide a method of treating a human withtransthyretin amyloidosis leading to cardiac amyloidosis andadministering to the human a therapeutically effective amount of acompound or composition as described herein. In certain embodiments, thetreatment reduces a symptom selected from the group consisting ofcongestive heart failure, cardiomegaly, dyspnea on exertion, peripheraledema, arrhythmias, palpitations, lightheadedness, syncope, depositionin the subendothelium of the peripheral vasculature can lead to severepostural hypotension, diastolic dysfunction, heart block, prematureventricular contractions, and various tachyarrhythmias.

Further embodiments provide a method of treating a human withtransthyretin amyloidosis leading to peripheral neuropathic disordersand administering to the human a therapeutically effective amount of acompound or composition as described herein. In certain embodiments, thetreatment reduces a symptom selected from the group consisting ofperipheral nerve problems, sensorimotor impairment, lower-limbneuropathy, upper-limb neuropathy, hyperalgesia, altered temperaturesensation, lower extremity weakness, pain, autonomic dysfunction, oftenmanifested as sexual or urinary dysfunction, symmetric sensoryimpairment and weakness, orthostatic hypotension, diarrhea, and/orimpotence.

Further embodiments provide a method of treating a human withtransthyretin amyloidosis leading to gastrointestinal disorders andadministering to the human a therapeutically effective amount of acompound or composition a described herein. In certain embodiments, thetreatment reduces a symptom selected from the group consisting ofdiarrhea, constipation, nausea, vomiting, and related kidney and liverdisorders.

Further provided is a method for reducing or preventing transthyretinamyloidosis comprising administering to a human a therapeuticallyeffective amount compound or composition as described herein, therebyreducing or preventing transthyretin amyloidosis.

Further provided is a method for reducing or preventing a cardiacdisease comprising administering to a human a therapeutically effectiveamount compound or composition as described herein, thereby reducing orpreventing a cardiac disease. Further provided is a method for reducingor preventing a neuropathic disease comprising administering to a humana therapeutically effective amount compound or composition as describedherein, thereby reducing or preventing a neuropathic disease. Furtherprovided is a method for reducing or preventing a gastrointestinaldisease comprising administering to a human a therapeutically effectiveamount compound or composition as described herein, thereby reducing orpreventing a gastrointestinal disease.

Further provided is a method for ameliorating a symptom of transthyretinamyloidosis, comprising administering to a human in need thereof acompound comprising a modified oligonucleotide consisting of 12 to 30linked nucleosides, wherein said modified oligonucleotide specificallyhybridizes to SEQ ID NO: 1, 2, or 4, thereby ameliorating a symptom oftransthyretin amyloidosis in the human.

Further provided is a method for reducing the rate of progression of asymptom associated with transthyretin amyloidosis, comprisingadministering to a human in need thereof a compound comprising amodified oligonucleotide consisting of 12 to 30 linked nucleosides,wherein said modified oligonucleotide specifically hybridizes to SEQ IDNO: 1, 2, or 4, thereby reducing the rate of progression a symptom oftransthyretin amyloidosis in the human.

Further provided is a method for reversing degeneration indicated by asymptom associated with a transthyretin amyloidosis, administering to ahuman in need thereof a compound comprising a modified oligonucleotideconsisting of 12 to 30 linked nucleosides, wherein said modifiedoligonucleotide specifically hybridizes to SEQ ID NO: 1, 2, or 4,thereby reversing degeneration indicated by a symptom of transthyretinamyloid disease in the human.

Further provided is a method for ameliorating a symptom of transthyretinamyloidosis, comprising administering to a human in need thereof acompound comprising a modified oligonucleotide consisting of 12 to 30linked nucleosides and having a nucleobase sequence comprising at least8 contiguous nucleobases of a nucleobase sequence recited in SEQ ID NO:80, thereby ameliorating a symptom of transthyretin amyloidosis in thehuman.

Further embodiments provide a method of treating a human withtransthyretin amyloidosis, administering to a human in need thereof acompound comprising a modified oligonucleotide consisting of 12 to 30linked nucleosides and having a nucleobase sequence comprising at least8 contiguous nucleobases of a nucleobase sequence recited in SEQ ID NO:80, thereby treating transthyretin amyloidosis in a human.

In certain embodiments, the symptom is a physical, cognitive,psychiatric, or peripheral symptom. In certain embodiments, the symptomis a physical symptom selected from the group consisting ofrestlessness, lack of coordination, nystagmus, spastic paraparesis, lackof muscle coordination, impaired vision, insomnia, unusual sensations,myoclonus, blindness, loss of speech, Carpal tunnel syndrome, seizures,subarachnoid hemorrhages, stroke and bleeding in the brain,hydrocephalus, ataxia, and spastic paralysis, coma, sensory neuropathy,parathesia, hypesthesia, motor neuropathy, autonomic neuropathy,orthostatic hypotension, cyclic constipation, cyclic diarrhea, nausea,vomiting, reduced sweating, impotence, delayed gastric emptying, urinaryretention, urinary incontinence, progressive cardiopathy, fatigue,shortness of breath, weight loss, lack of appetite, numbness, tingling,weakness, enlarged tongue, nephrotic syndrome, congestive heart failure,dyspnea on exertion, peripheral edema, arrhythmias, palpitations,light-headedness, syncope, postural hypotension, peripheral nerveproblems, sensory motor impairment, lower limb neuropathy, upper limbneuropathy, hyperalgesia, altered temperature sensation, lower extremityweakness, cachexia, peripheral edema, hepatomegaly, purpura, diastolicdysfunction, premature ventricular contractions, cranial neuropathy,diminished deep tendon reflexes, amyloid deposits in the corpus vitreum,vitreous opacity, dry eyes, glaucoma, scalloped appearance in thepupils, swelling of the feet due to water retention. In certainembodiments, the symptom is a cognitive symptom selected from the groupconsisting of impaired memory, impaired judgment, and thinking, impairedplanning, impaired flexibility, impaired abstract thinking, impairedrule acquisition, impaired initiation of appropriate actions, impairedinhibition of inappropriate actions, impaired short-term memory,impaired long-term memory, paranoia, disorientation, confusion,hallucination and dementia. In certain embodiments, the symptom is apsychiatric symptom selected from the group consisting of dementia;anxiety, depression, blunted affect, egocentrisms, aggression,compulsive behavior, irritability, personality changes, including,impaired memory, judgment, and thinking and suicidal ideation.

In certain embodiments the symptom is at least one of at least onephysical symptom, at least one cognitive symptom, at least onepsychiatric symptom, and at least one peripheral symptom.

In certain embodiments the physical symptom is selected from the groupconsisting of restlessness, lack of coordination, unintentionallyinitiated motions, unintentionally uncompleted motions, unsteady gait,chorea, rigidity, writhing motions, abnormal posturing, instability,abnormal facial expressions, difficulty chewing, difficulty swallowing,difficulty speaking, seizure, and sleep disturbances.

In certain embodiments the cognitive symptom is selected from the groupconsisting of impaired memory, impaired judgment, and thinking, impairedplanning, impaired flexibility, impaired abstract thinking, impairedrule acquisition, impaired initiation of appropriate actions, impairedinhibition of inappropriate actions, impaired short-term memory,impaired long-term memory, paranoia, disorientation, confusion,hallucination and dementia.

In certain embodiments the psychiatric symptom is selected from thegroup consisting of dementia; anxiety, depression, blunted affect,egocentrisms, aggression, compulsive behavior, irritability, personalitychanges, including, impaired memory, judgment, and thinking and suicidalideation.

In certain embodiments the peripheral symptom is selected from the groupconsisting of reduced brain mass, muscle atrophy, cardiac failure,impaired glucose tolerance, weight loss, osteoporosis, and testicularatrophy.

Also provided are methods and compounds for the preparation of amedicament for the treatment, prevention, or amelioration of a centralnervous system related disease.

Certain embodiments provide the use of a compound as described herein inthe manufacture of a medicament for treating, ameliorating, orpreventing a transthyretin amyloidosis.

Certain embodiments provide a compound as described herein for use intreating, preventing, or ameliorating transthyretin amyloidosis asdescribed herein by combination therapy with an additional agent ortherapy as described herein. Agents or therapies can be co-administeredor administered concomitantly.

Certain embodiments provide the use of a compound as described herein inthe manufacture of a medicament for treating, preventing, orameliorating transthyretin amyloidosis as described herein bycombination therapy with an additional agent or therapy as describedherein. Agents or therapies can be co-administered or administeredconcomitantly.

Certain embodiments provide the use of a compound as described herein inthe manufacture of a medicament for treating, preventing, orameliorating transthyretin amyloidosis as described herein in a patientwho is subsequently administered an additional agent or therapy asdescribed herein.

Certain embodiments provide a kit for treating, preventing, orameliorating transthyretin amyloidosis as described herein wherein thekit comprises:

-   (i) a compound as described herein; and alternatively-   (ii) an additional agent or therapy as described herein.

A kit as described herein may further include instructions for using thekit to treat, prevent, or ameliorate transthyretin amyloidosis asdescribed herein by combination therapy as described herein.

Antisense Compounds

Oligomeric compounds include, but are not limited to, oligonucleotides,oligonucleosides, oligonucleotide analogs, oligonucleotide mimetics,antisense compounds, antisense oligonucleotides, and siRNAs. Anoligomeric compound may be “antisense” to a target nucleic acid, meaningthat is capable of undergoing hybridization to a target nucleic acidthrough hydrogen bonding.

In certain embodiments, an antisense compound has a nucleobase sequencethat, when written in the 5′ to 3′ direction, comprises the reversecomplement of the target segment of a target nucleic acid to which it istargeted. In certain such embodiments, an antisense oligonucleotide hasa nucleobase sequence that, when written in the 5′ to 3′ direction,comprises the reverse complement of the target segment of a targetnucleic acid to which it is targeted.

In certain embodiments, an antisense compound targeted to atransthyretin nucleic acid is 12 to 30 nucleotides in length. In otherwords, antisense compounds are from 12 to 30 linked nucleobases. Inother embodiments, the antisense compound comprises a modifiedoligonucleotide consisting of 8 to 80, 12 to 50, 12 to 30, 15 to 30, 18to 24, 18 to 21, 19 to 22, or 20 linked nucleobases. In certain suchembodiments, the antisense compound comprises a modified oligonucleotideconsisting of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, or 80 linked nucleobases in length, or a range defined byany two of the above values.

In certain embodiments, the antisense compound comprises a shortened ortruncated modified oligonucleotide. The shortened or truncated modifiedoligonucleotide can have a single nucleoside deleted from the 5′ end (5′truncation), or alternatively from the 3′ end (3′ truncation). Ashortened or truncated oligonucleotide may have two nucleosides deletedfrom the 5′ end, or alternatively may have two subunits deleted from the3′ end. Alternatively, the deleted nucleosides may be dispersedthroughout the modified oligonucleotide, for example, in an antisensecompound having one nucleoside deleted from the 5′ end and onenucleoside deleted from the 3′ end.

When a single additional nucleoside is present in a lengthenedoligonucleotide, the additional nucleoside may be located at the 5′ or3′ end of the oligonucleotide. When two or more additional nucleosidesare present, the added nucleosides may be adjacent to each other, forexample, in an oligonucleotide having two nucleosides added to the 5′end (5′ addition), or alternatively to the 3′ end (3′ addition), of theoligonucleotide. Alternatively, the added nucleoside may be dispersedthroughout the antisense compound, for example, in an oligonucleotidehaving one nucleoside added to the 5′ end and one subunit added to the3′ end.

It is possible to increase or decrease the length of an antisensecompound, such as an antisense oligonucleotide, and/or introducemismatch bases without eliminating activity. For example, in Woolf etal. (Proc. Natl. Acad. Sci. USA 89:7305-7309, 1992), a series ofantisense oligonucleotides 13-25 nucleobases in length were tested fortheir ability to induce cleavage of a target RNA in an oocyte injectionmodel. Antisense oligonucleotides 25 nucleobases in length with 8 or 11mismatch bases near the ends of the antisense oligonucleotides were ableto direct specific cleavage of the target mRNA, albeit to a lesserextent than the antisense oligonucleotides that contained no mismatches.Similarly, target specific cleavage was achieved using 13 nucleobaseantisense oligonucleotides, including those with 1 or 3 mismatches.

Gautschi et al (J. Natl. Cancer Inst. 93:463-471, March 2001)demonstrated the ability of an oligonucleotide having 100%complementarity to the bcl-2 mRNA and having 3 mismatches to the bcl-xLmRNA to reduce the expression of both bcl-2 and bcl-xL in vitro and invivo. Furthermore, this oligonucleotide demonstrated potent anti-tumoractivity in vivo.

Maher and Dolnick (Nuc. Acid. Res. 16:3341-3358,1988) tested a series oftandem 14 nucleobase antisense oligonucleotides, and a 28 and 42nucleobase antisense oligonucleotides comprised of the sequence of twoor three of the tandem antisense oligonucleotides, respectively, fortheir ability to arrest translation of human DHFR in a rabbitreticulocyte assay. Each of the three 14 nucleobase antisenseoligonucleotides alone was able to inhibit translation, albeit at a moremodest level than the 28 or 42 nucleobase antisense oligonucleotides.

Antisense Compound Motifs

In certain embodiments, antisense compounds targeted to a transthyretinnucleic acid have chemically modified subunits arranged in patterns, ormotifs, to confer to the antisense compounds properties such as enhancedthe inhibitory activity, increased binding affinity for a target nucleicacid, or resistance to degradation by in vivo nucleases.

Chimeric antisense compounds typically contain at least one regionmodified so as to confer increased resistance to nuclease degradation,increased cellular uptake, increased binding affinity for the targetnucleic acid, and/or increased inhibitory activity. A second region of achimeric antisense compound may optionally serve as a substrate for thecellular endonuclease RNase H, which cleaves the RNA strand of anRNA:DNA duplex.

Antisense compounds having a gapmer motif are considered chimericantisense compounds. In a gapmer an internal region having a pluralityof nucleotides or linked nucleosides that supports RNaseH cleavage ispositioned between external regions having a plurality of nucleotides orlinked nucleosides that are chemically distinct from the nucleotides orlinked nucleosides of the internal region. In the case of an antisenseoligonucleotide having a gapmer motif, the gap segment generally servesas the substrate for endonuclease cleavage, while the wing segmentscomprise modified nucleosides. In certain embodiments, the regions of agapmer are differentiated by the types of sugar moieties comprising eachdistinct region. The types of sugar moieties that are used todifferentiate the regions of a gapmer may in some embodiments includeβ-D-ribonucleosides, β-D-deoxyribonucleosides, 2′-modified nucleosides(such 2′-modified nucleosides may include 2′-MOE, and 2′—O-CH₃, amongothers), and bicyclic sugar modified nucleosides (such bicyclic sugarmodified nucleosides may include those having a 4′—(CH₂)n-O—2′ bridge,where n=1 or n=2). Preferably, each distinct region comprises uniformsugar moieties. The wing-gap-wing motif is frequently described as“X-Y-Z”, where “X” represents the length of the 5′ wing region, “Y”represents the length of the gap region, and “Z” represents the lengthof the 3′ wing region. As used herein, a gapmer described as “X-Y-Z” hasa configuration such that the gap segment is positioned immediatelyadjacent each of the 5′ wing segment and the 3′ wing segment. Thus, nointervening nucleotides exist between the 5′ wing segment and gapsegment, or the gap segment and the 3′ wing segment. Any of theantisense compounds described herein can have a gapmer motif In someembodiments, X and Z are the same, in other embodiments they aredifferent. In a preferred embodiment, Y is between 8 and 15 nucleotides.X, Y or Z can be any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 25, 30 or more nucleotides. Thus, gapmersinclude, but are not limited to, for example 5-10-5, 4-8-4, 4-12-3,4-12-4, 3-14-3, 2-13-5, 2-16-2, 1-18-1, 3-10-3, 2-10-2, 1-10-1, 2-8-2,6-8-6 or 5-8-5.

In certain embodiments, the antisense compound as a “wingmer” motif,having a wing-gap or gap-wing configuration, i.e. an X-Y or Y-Zconfiguration as described above for the gapmer configuration. Thus,wingmer configurations include, but are not limited to, for example5-10, 8-4, 4-12, 12-4, 3-14, 16-2, 18-1, 10-3, 2-10, 1-10, 8-2, 2-13, or5-13.

In certain embodiments, antisense compounds targeted to a transthyretinnucleic acid possess a 5-10-5 gapmer motif.

In certain embodiments, antisense compounds targeted to a transthyretinnucleic acid possess a 6-8-6 gapmer motif.

In certain embodiments, antisense compounds targeted to a transthyretinnucleic acid possess a 5-8-5 gapmer motif.

In certain embodiments, an antisense compound targeted to atransthyretin nucleic acid has a gap-widened motif.

In certain embodiments, a gap-widened antisense oligonucleotide targetedto a transthyretin nucleic acid has a gap segment of ten2′-deoxyribonucleotides positioned immediately adjacent to and betweenwing segments of five chemically modified nucleosides. In certainembodiments, the chemical modification comprises a 2′-sugarmodification. In another embodiment, the chemical modification comprisesa 2′-MOE sugar modification.

In certain embodiments, a gap-widened antisense oligonucleotide targetedto a transthyretin nucleic acid has a gap segment of eight2′-deoxyribonucleotides positioned immediately adjacent to and betweenwing segments of five chemically modified nucleosides. . In certainembodiments, the chemical modification comprises a 2′-sugarmodification. In another embodiment, the chemical modification comprisesa 2′-MOE sugar modification.

In certain embodiments, a gap-widened antisense oligonucleotide targetedto a transthyretin nucleic acid has a gap segment of eight2′-deoxyribonucleotides positioned immediately adjacent to and betweenwing segments of six chemically modified nucleosides. In certainembodiments, the chemical modification comprises a 2′-sugarmodification. In another embodiment, the chemical modification comprisesa 2′-MOE sugar modification.

Target Nucleic Acids, Target Regions and Nucleotide Sequences

In certain embodiments, the transthyretin nucleic acid is any of thesequences set forth in GENBANK Accession No. NM_000371.2, firstdeposited with GENBANK® on Feb. 13, 2008 (incorporated herein as SEQ IDNO: 1), GENBANK Accession No. NT_010966.10 truncated from nucleotides2009236 to 2017289, first deposited with GENBANK® on Aug. 1, 2002(incorporated herein as SEQ ID NO: 2); exons 1-4 extracted from therhesus monkey genomic sequence GENBANK Accession No. NW_001105671.1,based on similarity to human exons; and GENBANK Accession No.NW_001105671.1 truncated from nucleotides 628000 to 638000 (incorporatedherein as SEQ ID NO: 4), first deposited with GENBANK® on Mar. 28, 2006.

It is understood that the sequence set forth in each SEQ ID NO in theExamples contained herein is independent of any modification to a sugarmoiety, an internucleoside linkage, or a nucleobase. As such, antisensecompounds defined by a SEQ ID NO may comprise, independently, one ormore modifications to a sugar moiety, an internucleoside linkage, or anucleobase. Antisense compounds described by Isis Number (Isis No) orISIS NO indicate a combination of nucleobase sequence and motif.

In certain embodiments, a target region is a structurally defined regionof the target nucleic acid. For example, a target region may encompass a3′ UTR, a 5′ UTR, an exon, an intron, an exon/intron junction, a codingregion, a translation initiation region, translation termination region,or other defined nucleic acid region. The structurally defined regionsfor transthyretin can be obtained by accession number from sequencedatabases such as NCBI and such information is incorporated herein byreference. In certain embodiments, a target region may encompass thesequence from a 5′ target site of one target segment within the targetregion to a 3′ target site of another target segment within the targetregion.

Targeting includes determination of at least one target segment to whichan antisense compound hybridizes, such that a desired effect occurs. Incertain embodiments, the desired effect is a reduction in mRNA targetnucleic acid levels. In certain embodiments, the desired effect isreduction of levels of protein encoded by the target nucleic acid or aphenotypic change associated with the target nucleic acid.

A target region may contain one or more target segments. Multiple targetsegments within a target region may be overlapping. Alternatively, theymay be non-overlapping. In certain embodiments, target segments within atarget region are separated by no more than about 300 nucleotides. Incertain embodiments, target segments within a target region areseparated by a number of nucleotides that is, is about, is no more than,is no more than about, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30,20, or 10 nucleotides on the target nucleic acid, or is a range definedby any two of the preceding values. In certain embodiments, targetsegments within a target region are separated by no more than, or nomore than about, 5 nucleotides on the target nucleic acid. In certainembodiments, target segments are contiguous. Contemplated are targetregions defined by a range having a starting nucleic acid that is any ofthe 5′ target sites or 3′ target sites listed herein.

Suitable target segments may be found within a 5′ UTR, a coding region,a 3′ UTR, an intron, an exon, or an exon/intron junction. Targetsegments containing a start codon or a stop codon are also suitabletarget segments. A suitable target segment may specifically exclude acertain structurally defined region such as the start codon or stopcodon.

The determination of suitable target segments may include a comparisonof the sequence of a target nucleic acid to other sequences throughoutthe genome. For example, the BLAST algorithm may be used to identifyregions of similarity amongst different nucleic acids. This comparisoncan prevent the selection of antisense compound sequences that mayhybridize in a non-specific manner to sequences other than a selectedtarget nucleic acid (i.e., non-target or off-target sequences).

There may be variation in activity (e.g., as defined by percentreduction of target nucleic acid levels) of the antisense compoundswithin an active target region. In certain embodiments, reductions intransthyretin mRNA levels are indicative of inhibition of transthyretinexpression. Reductions in levels of a transthyretin protein are alsoindicative of inhibition of target mRNA expression. Further, phenotypicchanges are indicative of inhibition of transthyretin expression. Forexample, increase in brain size to normal, improvement in motorcoordination, decrease in continual muscular spasms (dystonia), decreasein irritability and/or anxiety, improvement of memory, or an increase inenergy, among other phenotypic changes that may be assayed. Otherphenotypic indications, e.g., symptoms associated with transthyretinamyloidosis, may also be assessed as described below.

Hybridization

In some embodiments, hybridization occurs between an antisense compounddisclosed herein and a transthyretin nucleic acid. The most commonmechanism of hybridization involves hydrogen bonding (e.g.,Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding) betweencomplementary nucleobases of the nucleic acid molecules.

Hybridization can occur under varying conditions. Stringent conditionsare sequence-dependent and are determined by the nature and compositionof the nucleic acid molecules to be hybridized.

Methods of determining whether a sequence is specifically hybridizableto a target nucleic acid are well known in the art. In certainembodiments, the antisense compounds provided herein are specificallyhybridizable with a transthyretin nucleic acid.

Complementarity

An antisense compound and a target nucleic acid are complementary toeach other when a sufficient number of nucleobases of the antisensecompound can hydrogen bond with the corresponding nucleobases of thetarget nucleic acid, such that a desired effect will occur (e.g.,antisense inhibition of a target nucleic acid, such as a transthyretinnucleic acid).

An antisense compound may hybridize over one or more segments of atransthyretin nucleic acid such that intervening or adjacent segmentsare not involved in the hybridization event (e.g., a loop structure,mismatch or hairpin structure).

In certain embodiments, the antisense compounds provided herein, or aspecified portion thereof, are, or are at least, 70%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%complementary to a transthyretin nucleic acid, a target region, targetsegment, or specified portion thereof. Percent complementarity of anantisense compound with a target nucleic acid can be determined usingroutine methods.

For example, an antisense compound in which 18 of 20 nucleobases of theantisense compound are complementary to a target region, and wouldtherefore specifically hybridize, would represent 90 percentcomplementarity. In this example, the remaining noncomplementarynucleobases may be clustered or interspersed with complementarynucleobases and need not be contiguous to each other or to complementarynucleobases. As such, an antisense compound which is 18 nucleobases inlength having 4 (four) noncomplementary nucleobases which are flanked bytwo regions of complete complementarity with the target nucleic acidwould have 77.8% overall complementarity with the target nucleic acidand would thus fall within the scope of the present invention. Percentcomplementarity of an antisense compound with a region of a targetnucleic acid can be determined routinely using BLAST programs (basiclocal alignment search tools) and PowerBLAST programs known in the art(Altschul et al., J. Mol. Biol., 1990, 215, 403 410; Zhang and Madden,Genome Res., 1997, 7, 649 656). Percent homology, sequence identity orcomplementarity, can be determined by, for example, the Gap program(Wisconsin Sequence Analysis Package, Version 8 for Unix, GeneticsComputer Group, University Research Park, Madison Wis.), using defaultsettings, which uses the algorithm of Smith and Waterman (Adv. Appl.Math., 1981, 2, 482 489).

In certain embodiments, the antisense compounds provided herein, orspecified portions thereof, are fully complementary (i.e. 100%complementary) to a target nucleic acid, or specified portion thereof.For example, antisense compound may be fully complementary to atransthyretin nucleic acid, or a target region, or a target segment ortarget sequence thereof. As used herein, “fully complementary” meanseach nucleobase of an antisense compound is capable of precise basepairing with the corresponding nucleobases of a target nucleic acid. Forexample, a 20 nucleobase antisense compound is fully complementary to atarget sequence that is 400 nucleobases long, so long as there is acorresponding 20 nucleobase portion of the target nucleic acid that isfully complementary to the antisense compound. Fully complementary canalso be used in reference to a specified portion of the first and /orthe second nucleic acid. For example, a 20 nucleobase portion of a 30nucleobase antisense compound can be “fully complementary” to a targetsequence that is 400 nucleobases long. The 20 nucleobase portion of the30 nucleobase oligonucleotide is fully complementary to the targetsequence if the target sequence has a corresponding 20 nucleobaseportion wherein each nucleobase is complementary to the 20 nucleobaseportion of the antisense compound. At the same time, the entire 30nucleobase antisense compound may or may not be fully complementary tothe target sequence, depending on whether the remaining 10 nucleobasesof the antisense compound are also complementary to the target sequence.

The location of a non-complementary nucleobase may be at the 5′ end or3′ end of the antisense compound. Alternatively, the non-complementarynucleobase or nucleobases may be at an internal position of theantisense compound. When two or more non-complementary nucleobases arepresent, they may be contiguous (i.e. linked) or non-contiguous. In oneembodiment, a non-complementary nucleobase is located in the wingsegment of a gapmer antisense oligonucleotide.

In certain embodiments, antisense compounds that are, or are up to 12,13, 14, 15, 16, 17, 18, 19, or 20 nucleobases in length comprise no morethan 4, no more than 3, no more than 2, or no more than 1non-complementary nucleobase(s) relative to a target nucleic acid, suchas a transthyretin nucleic acid, or specified portion thereof.

In certain embodiments, antisense compounds that are, or are up to 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or30 nucleobases in length comprise no more than 6, no more than 5, nomore than 4, no more than 3, no more than 2, or no more than 1non-complementary nucleobase(s) relative to a target nucleic acid, suchas a transthyretin nucleic acid, or specified portion thereof.

The antisense compounds provided herein also include those which arecomplementary to a portion of a target nucleic acid. As used herein,“portion” refers to a defined number of contiguous (i.e. linked)nucleobases within a region or segment of a target nucleic acid. A“portion” can also refer to a defined number of contiguous nucleobasesof an antisense compound. In certain embodiments, the antisensecompounds, are complementary to at least an 8 nucleobase portion of atarget segment. In certain embodiments, the antisense compounds arecomplementary to at least a 12 nucleobase portion of a target segment.In certain embodiments, the antisense compounds are complementary to atleast a 15 nucleobase portion of a target segment. Also contemplated areantisense compounds that are complementary to at least a 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, or more nucleobase portion of a targetsegment, or a range defined by any two of these values.

Identity

The antisense compounds provided herein may also have a defined percentidentity to a particular nucleotide sequence, SEQ ID NO, or compoundrepresented by a specific Isis number, or portion thereof. As usedherein, an antisense compound is identical to the sequence disclosedherein if it has the same nucleobase pairing ability. For example, a RNAwhich contains uracil in place of thymidine in a disclosed DNA sequencewould be considered identical to the DNA sequence since both uracil andthymidine pair with adenine. Shortened and lengthened versions of theantisense compounds described herein as well as compounds havingnon-identical bases relative to the antisense compounds provided hereinalso are contemplated. The non-identical bases may be adjacent to eachother or dispersed throughout the antisense compound. Percent identityof an antisense compound is calculated according to the number of basesthat have identical base pairing relative to the sequence to which it isbeing compared.

In certain embodiments, the antisense compounds, or portions thereof,are at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%identical to one or more of the antisense compounds or SEQ ID NOs, or aportion thereof, disclosed herein.

Modifications

A nucleoside is a base-sugar combination. The nucleobase (also known asbase) portion of the nucleoside is normally a heterocyclic base moiety.Nucleotides are nucleosides that further include a phosphate groupcovalently linked to the sugar portion of the nucleoside. For thosenucleosides that include a pentofuranosyl sugar, the phosphate group canbe linked to the 2′, 3′ or 5′ hydroxyl moiety of the sugar.Oligonucleotides are formed through the covalent linkage of adjacentnucleosides to one another, to form a linear polymeric oligonucleotide.Within the oligonucleotide structure, the phosphate groups are commonlyreferred to as forming the internucleoside linkages of theoligonucleotide.

Modifications to antisense compounds encompass substitutions or changesto internucleoside linkages, sugar moieties, or nucleobases. Modifiedantisense compounds are often preferred over native forms because ofdesirable properties such as, for example, enhanced cellular uptake,enhanced affinity for nucleic acid target, increased stability in thepresence of nucleases, or increased inhibitory activity.

Chemically modified nucleosides may also be employed to increase thebinding affinity of a shortened or truncated antisense oligonucleotidefor its target nucleic acid. Consequently, comparable results can oftenbe obtained with shorter antisense compounds that have such chemicallymodified nucleosides.

Modified Internucleoside Linkages

The naturally occurring internucleoside linkage of RNA and DNA is a 3′to 5′ phosphodiester linkage. Antisense compounds having one or moremodified, i.e. non-naturally occurring, internucleoside linkages areoften selected over antisense compounds having naturally occurringinternucleoside linkages because of desirable properties such as, forexample, enhanced cellular uptake, enhanced affinity for target nucleicacids, and increased stability in the presence of nucleases.

Oligonucleotides having modified internucleoside linkages includeinternucleoside linkages that retain a phosphorus atom as well asinternucleoside linkages that do not have a phosphorus atom.Representative phosphorus containing internucleoside linkages include,but are not limited to, phosphodiesters, phosphotriesters,methylphosphonates, phosphoramidate, and phosphorothioates. Methods ofpreparation of phosphorous-containing and non-phosphorous-containinglinkages are well known.

In certain embodiments, antisense compounds targeted to a transthyretinnucleic acid comprise one or more modified internucleoside linkages. Incertain embodiments, the modified internucleoside linkages arephosphorothioate linkages. In certain embodiments, each internucleosidelinkage of an antisense compound is a phosphorothioate internucleosidelinkage.

Modified Sugar Moieties

Antisense compounds of the invention can optionally contain one or morenucleosides wherein the sugar group has been modified. Such sugarmodified nucleosides may impart enhanced nuclease stability, increasedbinding affinity, or some other beneficial biological property to theantisense compounds. In certain embodiments, nucleosides comprisechemically modified ribofuranose ring moieties. Examples of chemicallymodified ribofuranose rings include without limitation, addition ofsubstitutent groups (including 5′ and 2′ substituent groups, bridging ofnon-geminal ring atoms to form bicyclic nucleic acids (BNA), replacementof the ribosyl ring oxygen atom with S, N(R), or C(R₁)(R₂) (R, R₁ and R₂are each independently H, C₁-C₁₂ alkyl or a protecting group) andcombinations thereof. Examples of chemically modified sugars include2′—F—5′-methyl substituted nucleoside (see PCT International ApplicationWO 2008/101157 Published on Aug. 21, 2008 for other disclosed 5′,2′-bissubstituted nucleosides) or replacement of the ribosyl ring oxygen atomwith S with further substitution at the 2′-position (see published U.S.Patent Application US2005-0130923, published on Jun. 16, 2005) oralternatively 5′-substitution of a BNA (see PCT InternationalApplication WO 2007/134181 Published on Nov. 22, 2007 wherein LNA issubstituted with for example a 5′-methyl or a 5′-vinyl group).

Examples of nucleosides having modified sugar moieties include withoutlimitation nucleosides comprising 5′-vinyl, 5′-methyl (R or S), 4′—S,2′—F, 2′—OCH₃, 2′—OCH₂CH₃, 2′—OCH₂CH₂F and 2′—O(CH₂)₂OCH₃ substituentgroups. The substituent at the 2′ position can also be selected fromallyl, amino, azido, thio, O-allyl, O—C₁-C₁₀ alkyl, OCF₃, OCH₂F,O(CH₂)₂SCH₃, O(CH₂)₂—O—N(R_(m))(R_(a)), O—CH₂—C(═O)—N(R_(m))(R_(n)), andO—CH₂—C(═O)—N(R₁)—(CH₂)₂—N(R_(m))(R_(a)), where each R₁, R_(m) and R_(n)is, independently, H or substituted or unsubstituted C₁-C₁₀ alkyl.

As used herein, “bicyclic nucleosides” refer to modified nucleosidescomprising a bicyclic sugar moiety. Examples of bicyclic nucleosidesinclude without limitation nucleosides comprising a bridge between the4′ and the 2′ ribosyl ring atoms. In certain embodiments, antisensecompounds provided herein include one or more bicyclic nucleosidescomprising a 4′ to 2′ bridge. Examples of such 4′ to 2′ bridged bicyclicnucleosides, include but are not limited to one of the formulae:4′—(CH₂)—O—2′ (LNA); 4′—(CH₂)—S—2′; 4′—(CH₂)₂—O—2′ (ENA);4′-CH(CH₃)—O—2′ and 4′—CH(CH₂OCH₃)—O—2′ (and analogs thereof see U.S.Pat. No. 7,399,845, issued on Jul. 15, 2008); 4′—C(CH₃)(CH₃)—O—2′ (andanalogs thereof see published International Application WO/2009/006478,published Jan. 8, 2009); 4′—CH₂—N(OCH₃)—2′ (and analogs thereof seepublished International Application WO/2008/150729, published Dec. 11,2008); 4′—CH₂—O—N(CH₃)-2′ (see published U.S. Patent ApplicationUS2004-0171570, published Sep. 2, 2004); 4′—CH₂—N(R)—O—2′, wherein R isH, C₁-C₁₂ alkyl, or a protecting group (see U.S. Pat. 7,427,672, issuedon Sep. 23, 2008); 4′—CH₂—C(H)(CH₃)—2′ (see Chattopadhyaya et al., J.Org. Chem., 2009, 74, 118-134); and 4′—CH2-C—(═CH₂)—2′ (and analogsthereof see published International Application WO 2008/154401,published on Dec. 8, 2008).

Further reports related to bicyclic nucleosides can also be found inpublished literature (see for example: Singh et al., Chem. Commun.,1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630;Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 5633-5638;Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; Singh etal., J. Org. Chem., 1998, 63, 10035-10039; Srivastava et al., J. Am.Chem. Soc., 2007, 129(26) 8362-8379; Elayadi et al., Curr. OpinionInvest. Drugs, 2001, 2, 558-561; Braasch et al., Chem. Biol., 2001, 8,1-7; and Orum et al., Curr. Opinion Mol. Ther., 2001, 3, 239-243; U.S.Pat. Nos. 6,268,490; 6,525,191; 6,670,461; 6,770,748; 6,794,499;7,034,133; 7,053,207; 7,399,845; 7,547,684; and 7,696,345; U.S. PatentPublication No. US2008-0039618; US2009-0012281; U.S. Patent Ser. Nos.60/989,574; 61/026,995; 61/026,998; 61/056,564; 61/086,231; 61/097,787;and 61/099,844; Published PCT International applications WO 1994/014226;WO 2004/106356; WO 2005/021570; WO 2007/134181; WO 2008/150729; WO2008/154401; and WO 2009/006478. Each of the foregoing bicyclicnucleosides can be prepared having one or more stereochemical sugarconfigurations including for example α-L-ribofuranose andβ-D-ribofuranose (see PCT international application PCT/DK98/00393,published on Mar. 25, 1999 as WO 99/14226).

In certain embodiments, bicyclic sugar moieties of BNA nucleosidesinclude, but are not limited to, compounds having at least one bridgebetween the 4′ and the 2′ position of the pentofuranosyl sugar moietywherein such bridges independently comprises 1 or from 2 to 4 linkedgroups independently selected from —[C(R_(a))(R_(b))]_(n)—,—C(R_(a))═C(R_(b))—, —C(R_(a))═N—, —C(═O)—, —C(═NR_(a))—, —C(═S)—, —O—,—Si(R_(a))₂—, —S(═O)—, and —N(R_(a))—;

wherein:

x is 0, 1, or 2;

n is 1, 2, 3, or 4;

each R_(a) and R_(b) is, independently, H, a protecting group, hydroxyl,C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substitutedC₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl,substituted C₅-C₂₀ aryl, heterocycle radical, substituted heterocycleradical, heteroaryl, substituted heteroaryl, C₅-C₇ alicyclic radical,substituted C₅-C₇ alicyclic radical, halogen, OJ₁, NJ₁J₂, SJ₁, N₃,COOJ₁, acyl (C(═O)—H), substituted acyl, CN, sulfonyl (S(═O)₂-J₁), orsulfoxyl (S(═O)-J₁); and

each J₁ and J₂ is, independently, H, C₁-C₁₂ alkyl, substituted C₁-C₁₂alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl,substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl, substituted C₅-C₂₀ aryl, acyl(C(═O)—H), substituted acyl, a heterocycle radical, a substitutedheterocycle radical, C₁-C₁₂ aminoalkyl, substituted C₁-C₁₂ aminoalkyl ora protecting group.

In certain embodiments, the bridge of a bicyclic sugar moiety is—[C(R_(a))(R_(b))]_(n)—, —[C(R_(a))(R_(b))]_(n)—O—,—C(R_(a)R_(b))—N(R)—O— or —C(R_(a)R_(b))—O—N(R)—. In certainembodiments, the bridge is 4′—CH₂—2′, 4′—(CH₂)₂—2′,4′—(CH₂)₃—2′,4′—CH₂—O—2′,4′—(CH₂)₂—O—2′,4′—CH₂—O—N(R)—2′ and4′—CH₂—N(R)—O—2′— wherein each R is, independently, H, a protectinggroup or C₁-C₁₂ alkyl.

In certain embodiments, bicyclic nucleosides are further defined byisomeric configuration. For example, a nucleoside comprising a 4′-2′methylene-oxy bridge, may be in the α-L configuration or in the β-Dconfiguration. Previously, α-L-methyleneoxy (4′—CH₂—O—2′) BNA's havebeen incorporated into antisense oligonucleotides that showed antisenseactivity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372).

In certain embodiments, bicyclic nucleosides include, but are notlimited to, (A) α-L-methyleneoxy (4′—CH₂—O—2′) BNA, (B) β-D-methyleneoxy(4′—CH₂—O—2′) BNA, (C) ethyleneoxy (4′—(CH₂)₂—O—2′) BNA, (D) aminooxy(4′—CH₂—O—N(R)—2′) BNA, (E) oxyamino (4′—CH₂—N(R)—O—2′) BNA, and (F)methyl(methyleneoxy) (4′—CH(CH₃)—O—2′) BNA, (G) methylene-thio(4′—CH₂—S—2′) BNA, (H) methylene-amino (4′—CH₂—N(R)—2′) BNA, (I) methylcarbocyclic (4′—CH₂—CH(CH₃)—2′) BNA, and (J) propylene carbocyclic(4′—(CH₂)₃—2′) BNA as depicted below.

wherein Bx is the base moiety and R is independently H, a protectinggroup or C₁-C₁₂ alkyl.

In certain embodiments, bicyclic nucleosides are provided having FormulaI:

wherein:

Bx is a heterocyclic base moiety;

-Q_(a)-Q_(b)-Q_(c)- is —CH₂—N(R_(c))—CH₂—, —C(═O)—N(R_(c))—CH₂—,—CH₂—O—N(R_(c))—, —CH₂—N(R_(c))—O— or —N(R_(c))—O—CH₂;

R_(c) is C₁-C₁₂ alkyl or an amino protecting group; and

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety or acovalent attachment to a support medium.

In certain embodiments, bicyclic nucleosides are provided having FormulaII:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety or acovalent attachment to a support medium;

Z_(a) is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₁-C₆alkyl, substituted C₂-C₆ alkenyl, substituted C₂-C₆ alkynyl, acyl,substituted acyl, substituted amide, thiol or substituted thio.

In one embodiment, each of the substituted groups is, independently,mono or poly substituted with substituent groups independently selectedfrom halogen, oxo, hydroxyl, OJ_(c), NJ_(c)J_(d), SJ_(c), N₃,OC(═X)J_(c), and NJ_(c)C(═X)NJ_(c)J_(d), wherein each J_(c), J_(d) andJ_(e) is, independently, H, C₁-C₆ alkyl, or substituted C₁-C₆ alkyl andX is O or NJ_(c).

In certain embodiments, bicyclic nucleosides are provided having FormulaIII:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety or acovalent attachment to a support medium;

Z_(b) is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₁-C₆alkyl, substituted C₂-C₆ alkenyl, substituted C₂-C₆ alkynyl orsubstituted acyl (C(═O)—).

In certain embodiments, bicyclic nucleosides are provided having FormulaIV:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety or acovalent attachment to a support medium;

R_(d) is C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl,substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl;

each q_(a), q_(b), q_(c) and q_(d) is, independently, H, halogen, C₁-C₆alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl, C₁-C₆ alkoxyl,substituted C₁-C₆ alkoxyl, acyl, substituted acyl, C₁-C₆ aminoalkyl orsubstituted C₁-C₆ aminoalkyl;

In certain embodiments, bicyclic nucleosides are provided having FormulaV:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety or acovalent attachment to a support medium;

q_(a), q_(b), q_(e) and q_(f) are each, independently, hydrogen,halogen, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl,substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl,C₁-C₁₂ alkoxy, substituted C₁-C₁₂ alkoxy, OJ_(j), SJ_(j), SOJ₂J_(j),NJ_(j)J_(k), N₃, CN, C(═O)OJ_(j), C(═O)NJ_(j)J_(k), C(═O)J_(j),O—C(═O)NJ_(j)J_(k), N(H)C(═NH)NJ_(j)J_(k), N(H)C(═O)NJ_(j)J_(k), orN(H)C(═S)NJ_(j)J_(k);

or q_(e) and q_(f) together are ═C(q_(g))(q_(h));

q_(g) and q_(h) are each, independently, H, halogen, C₁-C₁₂ alkyl orsubstituted C₁-C₁₂ alkyl.

The synthesis and preparation of the methyleneoxy (4′—CH₂—O—2′) BNAmonomers adenine, cytosine, guanine, 5-methyl-cytosine, thymine anduracil, along with their oligomerization, and nucleic acid recognitionproperties have been described (Koshkin et al., Tetrahedron, 1998, 54,3607-3630). BNAs and preparation thereof are also described in WO98/39352 and WO 99/14226.

Analogs of methyleneoxy (4′—CH₂—O—2′) BNA and 2′-thio-BNAs, have alsobeen prepared (Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8,2219-2222). Preparation of locked nucleoside analogs comprisingoligodeoxyribonucleotide duplexes as substrates for nucleic acidpolymerases has also been described (Wengel et al., WO 99/14226).Furthermore, synthesis of 2′-amino-BNA, a novel comformationallyrestricted high-affinity oligonucleotide analog has been described inthe art (Singh et al., J. Org. Chem., 1998, 63, 10035-10039). Inaddition, 2′-amino- and 2′-methylamino-BNA's have been prepared and thethermal stability of their duplexes with complementary RNA and DNAstrands has been previously reported.

In certain embodiments, bicyclic nucleosides are provided having FormulaVI:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety or acovalent attachment to a support medium;

each q_(i), q_(j), q_(k) and q_(l) is, independently, H, halogen, C₁-C₁₂alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₁-C₁₂ alkoxyl,substituted C₁-C₁₂ alkoxyl, OJ_(j), SJ_(j), SOJ_(j), SO₂J_(j),NJ_(j)J_(k), N₃, CN, C(═O)OJ_(j), C(═O)NJ_(j)J_(k), C(═O)J_(k),O—C(═O)NJ_(j)J_(k), N(H)C(═NH)NJ_(j)J_(k), N(H)C(═O)NJ_(j)J_(k) orN(H)C(═S)NJ_(j)J_(k); and

q_(i) and q_(j) or q_(i) and q_(k) together are ═C(q_(g))(q_(h)),wherein q_(g) and q_(h) are each, independently, H, halogen, C₁-C₁₂alkyl or substituted C₁-C₁₂ alkyl.

One carbocyclic bicyclic nucleoside having a 4′—(CH₂)₃—2′ bridge and thealkenyl analog bridge 4′—CH═CH—CH₂—2′ have been described (Freier etal., Nucleic Acids Research, 1997, 25(22), 4429-4443 and Albaek et al.,J. Org. Chem., 2006, 71, 7731-7740). The synthesis and preparation ofcarbocyclic bicyclic nucleosides along with their oligomerization andbiochemical studies have also been described (Srivastava et al., J. Am.Chem. Soc., 2007, 129(26), 8362-8379).

As used herein, “4′-2′ bicyclic nucleoside” or “4′ to 2′ bicyclicnucleoside” refers to a bicyclic nucleoside comprising a furanose ringcomprising a bridge connecting two carbon atoms of the furanose ringconnects the 2′ carbon atom and the 4′ carbon atom of the sugar ring.

As used herein, “monocylic nucleosides” refer to nucleosides comprisingmodified sugar moieties that are not bicyclic sugar moieties. In certainembodiments, the sugar moiety, or sugar moiety analogue, of a nucleosidemay be modified or substituted at any position.

As used herein, “2′-modified sugar” means a furanosyl sugar modified atthe 2′ position. In certain embodiments, such modifications includesubstituents selected from: a halide, including, but not limited tosubstituted and unsubstituted alkoxy, substituted and unsubstitutedthioalkyl, substituted and unsubstituted amino alkyl, substituted andunsubstituted alkyl, substituted and unsubstituted allyl, andsubstituted and unsubstituted alkynyl. In certain embodiments, 2′modifications are selected from substituents including, but not limitedto: O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)F,O(CH₂)_(n)ONH₂, OCH₂C(═O)N(H)CH₃, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃]₂, wheren and m are from 1 to about 10. Other 2′-substituent groups can also beselected from: C₁-C₁₂ alkyl, substituted alkyl, alkenyl, alkynyl,alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, F,CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl,heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl,an RNA cleaving group, a reporter group, an intercalator, a group forimproving pharmacokinetic properties, or a group for improving thepharmacodynamic properties of an antisense compound, and othersubstituents having similar properties. In certain embodiments, modifednucleosides comprise a 2′-MOE side chain (Baker et al., J. Biol. Chem.,1997, 272, 11944-12000). Such 2′-MOE substitution have been described ashaving improved binding affinity compared to unmodified nucleosides andto other modified nucleosides, such as 2′-O-methyl, O-propyl, andO-aminopropyl. Oligonucleotides having the 2′-MOE substituent also havebeen shown to be antisense inhibitors of gene expression with promisingfeatures for in vivo use (Martin, Helv. Chim. Acta, 1995, 78, 486-504;Altmann et al., Chimia, 1996, 50, 168-176; Altmann et al., Biochem. Soc.Trans., 1996, 24, 630-637; and Altmann et al., Nucleosides Nucleotides,1997, 16, 917-926).

As used herein, a “modified tetrahydropyran nucleoside” or “modified THPnucleoside” means a nucleoside having a six-membered tetrahydropyran“sugar” substituted in for the pentofuranosyl residue in normalnucleosides (a sugar surrogate). Modified THP nucleosides include, butare not limited to, what is referred to in the art as hexitol nucleicacid (HNA), anitol nucleic acid (ANA), manitol nucleic acid (MNA) (seeLeumann, Bioorg. Med. Chem., 2002, 10, 841-854), fluoro HNA (F-HNA) orthose compounds having Formula VII:

wherein independently for each of said at least one tetrahydropyrannucleoside analog of Formula VII:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently, an internucleoside linkinggroup linking the tetrahydropyran nucleoside analog to the antisensecompound or one of T_(a) and T_(b) is an internucleoside linking grouplinking the tetrahydropyran nucleoside analog to the antisense compoundand the other of T_(a) and T_(b) is H, a hydroxyl protecting group, alinked conjugate group or a 5′ or 3′-terminal group;

q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each independently, H, C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆alkynyl or substituted C₂-C₆ alkynyl; and each of R₁ and R₂ is selectedfrom hydrogen, hydroxyl, halogen, subsitituted or unsubstituted alkoxy,NJ₁J₂, SJ₁, N₃, OC(═X)J₁, OC(═X)NJ₁J₂, NJ₃C(═X)NJ₁J₂ and CN, wherein Xis O, S or NJ₁ and each J₁, J₂ and J₃ is, independently, H or C₁-C₆alkyl.

In certain embodiments, the modified THP nucleosides of Formula VII areprovided wherein q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each H. In certainembodiments, at least one of q₁, q₂, q₃, q₄, q₅, q₆ and q₇ is other thanH. In certain embodiments, at least one of q₁, q₂, q₃, q₄, q₅, q₆ and q₇is methyl. In certain embodiments, THP nucleosides of Formula VII areprovided wherein one of R₁ and R₂ is fluoro. In certain embodiments, R₁is fluoro and R₂ is H; R₁ is methoxy and R₂ is H, and R₁ is H and R₂ ismethoxyethoxy.

As used herein, “2′-modified” or “2′-substituted” refers to a nucleosidecomprising a sugar comprising a substituent at the 2′ position otherthan H or OH. 2′-modified nucleosides, include, but are not limited to,bicyclic nucleosides wherein the bridge connecting two carbon atoms ofthe sugar ring connects the 2′ carbon and another carbon of the sugarring; and nucleosides with non-bridging 2′ substituents, such as allyl,amino, azido, thio, O-allyl, O—C₁-C₁₀ alkyl, —OCF₃, O—(CH₂)₂—O—CH₃,2′—O—(CH₂)₂SCH₃, —O—(CH₂)₂—O—N(R_(m))(R_(n)), orO—CH₂—C(═O)—N(R_(m))(R_(n)), where each R_(m) and R_(n) is,independently, H or substituted or unsubstituted C₁-C₁₀ alkyl.2′-modifed nucleosides may further comprise other modifications, forexample at other positions of the sugar and/or at the nucleobase.

As used herein, “2′-F” refers to a nucleoside comprising a sugarcomprising a fluoro group at the 2′ position.

As used herein, “2′—OMe” or “2′—OCH₃” or “2′—O-methyl” each refers to anucleoside comprising a sugar comprising an —OCH₃ group at the 2′position of the sugar ring.

As used herein, “MOE” or “2′-MOE” or “2′—OCH₂CH₂OCH₃” or“2′—O-methoxyethyl” each refers to a nucleoside comprising a sugarcomprising a -OCH2CH2OCH3 group at the 2′ position of the sugar ring.

As used herein, “oligonucleotide” refers to a compound comprising aplurality of linked nucleosides. In certain embodiments, one or more ofthe plurality of nucleosides is modified. In certain embodiments, anoligonucleotide comprises one or more ribonucleosides (RNA) and/ordeoxyribonucleosides (DNA).

Many other bicyclo and tricyclo sugar surrogate ring systems are alsoknown in the art that can be used to modify nucleosides forincorporation into antisense compounds (see for example review article:Leumann, Bioorg. Med. Chem., 2002, 10, 841-854).

-   Such ring systems can undergo various additional substitutions to    enhance activity.

Methods for the preparations of modified sugars are well known to thoseskilled in the art.

In nucleotides having modified sugar moieties, the nucleobase moieties(natural, modified or a combination thereof) are maintained forhybridization with an appropriate nucleic acid target.

In certain embodiments, antisense compounds comprise one or morenucleosides having modified sugar moieties. In certain embodiments, themodified sugar moiety is 2′-MOE. In certain embodiments, the 2′-MOEmodified nucleosides are arranged in a gapmer motif In certainembodiments, the modified sugar moiety is a bicyclic nucleoside having a(4′—CH(CH₃)—O—2′) bridging group. In certain embodiments, the(4′—CH(CH₃)—O—2′) modified nucleosides are arranged throughout the wingsof a gapmer motif.

Modified Nucleobases

Nucleobase (or base) modifications or substitutions are structurallydistinguishable from, yet functionally interchangeable with, naturallyoccurring or synthetic unmodified nucleobases. Both natural and modifiednucleobases are capable of participating in hydrogen bonding. Suchnucleobase modifications may impart nuclease stability, binding affinityor some other beneficial biological property to antisense compounds.Modified nucleobases include synthetic and natural nucleobases such as,for example, 5-methylcytosine (5-mc-C). Certain nucleobasesubstitutions, including 5-methylcytosine substitutions, areparticularly useful for increasing the binding affinity of an antisensecompound for a target nucleic acid. For example, 5-methylcytosinesubstitutions have been shown to increase nucleic acid duplex stabilityby 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds.,Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp.276-278).

Additional unmodified nucleobases include 5-hydroxymethyl cytosine,xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,5-halouracil and cytosine, 5-propynyl (—C═C—CH₃) uracil and cytosine andother alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosineand thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino,8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines andguanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and3-deazaadenine.

Heterocyclic base moieties may also include those in which the purine orpyrimidine base is replaced with other heterocycles, for example7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone.Nucleobases that are particularly useful for increasing the bindingaffinity of antisense compounds include 5-substituted pyrimidines,6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.

In certain embodiments, antisense compounds targeted to a transthyretinnucleic acid comprise one or more modified nucleobases. In certainembodiments, gap-widened antisense oligonucleotides targeted to atransthyretin nucleic acid comprise one or more modified nucleobases. Incertain embodiments, the modified nucleobase is 5-methylcytosine. Incertain embodiments, each cytosine is a 5-methylcytosine.

Compositions and Methods For Formulating Pharmaceutical Compositions

Antisense oligonucleotides may be admixed with pharmaceuticallyacceptable active or inert substance for the preparation ofpharmaceutical compositions or formulations. Compositions and methodsfor the formulation of pharmaceutical compositions are dependent upon anumber of criteria, including, but not limited to, route ofadministration, extent of disease, or dose to be administered.

Antisense compound targeted to a transthyretin nucleic acid can beutilized in pharmaceutical compositions by combining the antisensecompound with a suitable pharmaceutically acceptable diluent or carrier.A pharmaceutically acceptable diluent includes phosphate-buffered saline(PBS). PBS is a diluent suitable for use in compositions to be deliveredparenterally. Accordingly, in one embodiment, employed in the methodsdescribed herein is a pharmaceutical composition comprising an antisensecompound targeted to a transthyretin nucleic acid and a pharmaceuticallyacceptable diluent. In certain embodiments, the pharmaceuticallyacceptable diluent is PBS. In certain embodiments, the antisensecompound is an antisense oligonucleotide.

Pharmaceutical compositions comprising antisense compounds encompass anypharmaceutically acceptable salts, esters, or salts of such esters, orany other oligonucleotide which, upon administration to an animal,including a human, is capable of providing (directly or indirectly) thebiologically active metabolite or residue thereof. Accordingly, forexample, the disclosure is also drawn to pharmaceutically acceptablesalts of antisense compounds, prodrugs, pharmaceutically acceptablesalts of such prodrugs, and other bioequivalents. Suitablepharmaceutically acceptable salts include, but are not limited to,sodium and potassium salts.

A prodrug can include the incorporation of additional nucleosides at oneor both ends of an antisense compound which are cleaved by endogenousnucleases within the body, to form the active antisense compound.

Conjugated Antisense Compounds

Antisense compounds may be covalently linked to one or more moieties orconjugates which enhance the activity, cellular distribution or cellularuptake of the resulting antisense oligonucleotides. Typical conjugategroups include cholesterol moieties and lipid moieties. Additionalconjugate groups include carbohydrates, phospholipids, biotin,phenazine, folate, phenanthridine, anthraquinone, acridine,fluoresceins, rhodamines, coumarins, and dyes.

Antisense compounds can also be modified to have one or more stabilizinggroups that are generally attached to one or both termini of antisensecompounds to enhance properties such as, for example, nucleasestability. Included in stabilizing groups are cap structures. Theseterminal modifications protect the antisense compound having terminalnucleic acid from exonuclease degradation, and can help in deliveryand/or localization within a cell. The cap can be present at the5′-terminus (5′-cap), or at the 3-terminus (3′-cap), or can be presenton both termini. Cap structures are well known in the art and include,for example, inverted deoxy abasic caps. Further 3′ and 5′-stabilizinggroups that can be used to cap one or both ends of an antisense compoundto impart nuclease stability include those disclosed in WO 03/004602published on Jan. 16, 2003.

Cell Culture and Antisense Compounds Treatment

The effects of antisense compounds on the level, activity or expressionof transthyretin nucleic acids can be tested in vitro in a variety ofcell types. Cell types used for such analyses are available fromcommercial vendors (e.g. American Type Culture Collection, Manassus,Va.; Zen-Bio, Inc., Research Triangle Park, N.C.; Clonetics Corporation,Walkersville, Md.) and cells are cultured according to the vendor'sinstructions using commercially available reagents (e.g. Invitrogen LifeTechnologies, Carlsbad, Calif.). Illustrative cell types include, butare not limited to, HepG2 cells, Hep3B cells, primary hepatocytes, A549cells, GM04281 fibroblasts and LLC-MK2 cells.

In Vitro Testing of Antisense Oligonucleotides

Described herein are methods for treatment of cells with antisenseoligonucleotides, which can be modified appropriately for treatment withother antisense compounds.

In general, cells are treated with antisense oligonucleotides when thecells reach approximately 60-80% confluence in culture.

One reagent commonly used to introduce antisense oligonucleotides intocultured cells includes the cationic lipid transfection reagentLIPOFECTIN® (Invitrogen, Carlsbad, Calif.). Antisense oligonucleotidesare mixed with LIPOFECTIN® in OPTI-MEM® 1 (Invitrogen, Carlsbad, Calif.)to achieve the desired final concentration of antisense oligonucleotideand a LIPOFECTIN® concentration that typically ranges 2 to 12 ug/mL per100 nM antisense oligonucleotide.

Another reagent used to introduce antisense oligonucleotides intocultured cells includes LIPOFECTAMINE 2000® (Invitrogen, Carlsbad,Calif.). Antisense oligonucleotide is mixed with LIPOFECTAMINE 2000® inOPTI-MEM® 1 reduced serum medium (Invitrogen, Carlsbad, Calif.) toachieve the desired concentration of antisense oligonucleotide and aLIPOFECTAMINE® concentration that typically ranges 2 to 12 ug/mL per 100nM antisense oligonucleotide.

Another reagent used to introduce antisense oligonucleotides intocultured cells includes Cytofectin® (Invitrogen, Carlsbad, Calif.).Antisense oligonucleotide is mixed with Cytofectin® in OPTI-MEM® 1reduced serum medium (Invitrogen, Carlsbad, Calif.) to achieve thedesired concentration of antisense oligonucleotide and a Cytofectin®concentration that typically ranges 2 to 12 ug/mL per 100 nM antisenseoligonucleotide.

Another technique used to introduce antisense oligonucleotides intocultured cells includes electroporation.

Cells are treated with antisense oligonucleotides by routine methods.Cells are typically harvested 16-24 hours after antisenseoligonucleotide treatment, at which time RNA or protein levels of targetnucleic acids are measured by methods known in the art and describedherein. In general, when treatments are performed in multiplereplicates, the data are presented as the average of the replicatetreatments.

The concentration of antisense oligonucleotide used varies from cellline to cell line. Methods to determine the optimal antisenseoligonucleotide concentration for a particular cell line are well knownin the art. Antisense oligonucleotides are typically used atconcentrations ranging from 1 nM to 300 nM when transfected withLIPOFECTAMINE2000®, Lipofectin or Cytofectin. Antisense oligonucleotidesare used at higher concentrations ranging from 625 to 20,000 nM whentransfected using electroporation.

RNA Isolation

RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA.Methods of RNA isolation are well known in the art. RNA is preparedusing methods well known in the art, for example, using the TRIZOL®Reagent (Invitrogen, Carlsbad, Calif.) according to the manufacturer'srecommended protocols.

Analysis of Inhibition of Target Levels or Expression

Inhibition of levels or expression of a transthyretin nucleic acid canbe assayed in a variety of ways known in the art. For example, targetnucleic acid levels can be quantitated by, e.g., Northern blot analysis,competitive polymerase chain reaction (PCR), or quantitaive real-timePCR. RNA analysis can be performed on total cellular RNA or poly(A)+mRNA. Methods of RNA isolation are well known in the art. Northern blotanalysis is also routine in the art. Quantitative real-time PCR can beconveniently accomplished using the commercially available ABI PRISM®7600, 7700, or 7900 Sequence Detection System, available from PE-AppliedBiosystems, Foster City, Calif. and used according to manufacturer'sinstructions.

Quantitative Real-Time PCR Analysis of Target RNA Levels

Quantitation of target RNA levels may be accomplished by quantitativereal-time PCR using the ABI PRISM® 7600, 7700, or 7900 SequenceDetection System (PE-Applied Biosystems, Foster City, Calif.) accordingto manufacturer's instructions. Methods of quantitative real-time PCRare well known in the art.

Prior to real-time PCR, the isolated RNA is subjected to a reversetranscriptase (RT) reaction, which produces complementary DNA (cDNA)that is then used as the substrate for the real-time PCR amplification.The RT and real-time PCR reactions are performed sequentially in thesame sample well. RT and real-time PCR reagents are obtained fromInvitrogen (Carlsbad, Calif.). RT, real-time-PCR reactions are carriedout by methods well known to those skilled in the art.

Gene (or RNA) target quantities obtained by real time PCR are normalizedusing either the expression level of a gene whose expression isconstant, such as cyclophilin A, or by quantifying total RNA usingRIBOGREEN® (Invitrogen, Inc. Carlsbad, Calif.). Cyclophilin A expressionis quantified by real time PCR, by being run simultaneously with thetarget, multiplexing, or separately. Total RNA is quantified usingRIBOGREEN® RNA quantification reagent (Invitrogen, Inc. Eugene, Oreg.).Methods of RNA quantification by RIBOGREEN® are taught in Jones, L. J.,et al, (Analytical Biochemistry, 1998, 265, 368-374). A CYTOFLUOR® 4000instrument (PE Applied Biosystems) is used to measure RIBOGREEN®fluorescence.

Probes and primers are designed to hybridize to a transthyretin nucleicacid. Methods for designing real-time PCR probes and primers are wellknown in the art, and may include the use of software such as PRIMEREXPRESS® Software (Applied Biosystems, Foster City, Calif.).

Analysis of Protein Levels

Antisense inhibition of transthyretin nucleic acids can be assessed bymeasuring transthyretin protein levels. Protein levels of transthyretincan be evaluated or quantitated in a variety of ways well known in theart, such as immunoprecipitation, Western blot analysis(immunoblotting), enzyme-linked immunosorbent assay (ELISA),quantitative protein assays, protein activity assays (for example,caspase activity assays), immunohistochemistry, immunocytochemistry orfluorescence-activated cell sorting (FACS). Antibodies directed to atarget can be identified and obtained from a variety of sources, such asthe MSRS catalog of antibodies (Aerie Corporation, Birmingham, Mich.),or can be prepared via conventional monoclonal or polyclonal antibodygeneration methods well known in the art. Antibodies useful for thedetection of human and rat transthyretin are commercially available.

In Vivo Testing of Antisense Compounds

Antisense compounds, for example, antisense oligonucleotides, are testedin animals to assess their ability to inhibit expression oftransthyretin and produce phenotypic changes. Testing may be performedin normal animals, or in experimental disease models. For administrationto animals, antisense oligonucleotides are formulated in apharmaceutically acceptable diluent, such as phosphate-buffered saline.Administration includes parenteral routes of administration. Following aperiod of treatment with antisense oligonucleotides, RNA is isolatedfrom tissue and changes in transthyretin nucleic acid expression aremeasured. Changes in transthyretin protein levels are also measured.

Certain Compounds

About two hundred and forty six newly designed antisense compounds ofvarious lengths, motifs and backbone composition were tested for theireffect on human transthyretin mRNA in vitro in several cell types. Thenew compounds were compared with about seventy nine previously designedcompounds including ISIS NOs. 304267, 304268, 304280, 304284, 304285,304286, 304287, 304288, 304289, 304290, 304291, 304292, 304293, 304294,304296, 304297, 304298, 304299, 304300, 304301, 304302, 304303, 304304,304307, 304308, 304309, 304311, and 304312 which have previously beendetermined to be some of the most potent antisense compounds in vitro(see e.g., U.S. Patent Publication Nos. US2005/0244869 andUS2009/0082300). Of the about three hundred and twenty five newlydesigned and previously designed antisense compounds, about fifteencompounds were selected for further study based on in vitro potency. Theselected compounds were tested for in vivo potency and tolerability in atransgenic mouse model (see Example 10). Of the fifteen compoundstested, eleven were selected and tested for systemic tolerability (seeExample 11) and half-life measurement in liver (see Example 12) in CD1mice, and also for systemic tolerability (see Example 13) andpharmacokinetic studies of oligonucleotide concentration in liver (seeExample 14) in Sprague-Dawley rats. From these studies, seven compoundswere tested for dose dependent inhibition and tolerability in transgenicmice (see Example 15). Furthermore, fifteen additional compounds wereselected from Table 1 and six additional compounds with various motifswere designed with overlapping sequences to ISIS 420951, which displayedhigh potency and tolerability in the above-mentioned assays. Theseadditional compounds were compared with ISIS 420951 for potency andtolerability in transgenic mice (see Example 16). Based on all thesestudies (Examples 10-16), twenty two compounds were selected and testedfor systemic tolerability in CD1 mice (see Example 17). Seven compoundswere considered tolerable in the mouse model and further tested forsystemic tolerability in Sprague-Dawley rats (see Example 18) and forpharmacokinetic studies of oligonucleotide concentration in the liverand kidney (see Example 19). The seven compounds were also tested fordose-dependent potency in transgenic mice (see Example 20).

Final evaluation of these studies (Examples 16-20), led to the selectionof nine compounds having a nucleobase sequence of a sequence recited inSEQ ID NO: 25, 78, 80, 86, 87, 115, 120, 122 and 124. By virtue of theircomplementary sequence, the compounds are complementary to the regions505-524, 507-526, 508-527, 513-532, 515-534, 516-535, 580-599, 585-604,587-606, or 589-608 of SEQ ID NO: 1. In certain embodiments, thecompounds targeting the listed regions, as further described herein,comprise a modified oligonucleotide having some nucleobase portion ofthe sequence recited in the SEQ ID NOs, as further described herein, Incertain embodiments, the compounds targeting the listed regions orhaving a nucleobase portion of a sequence recited in the listed SEQ IDNOs can be of various length, as further described herein, and can haveone of various motifs, as further described herein. In certainembodiments, a compound targeting a region or having a nucleobaseportion of a sequence recited in the listed SEQ ID NOs has the specificlength and motif as indicated by the ISIS NOs: ISIS 304299, ISIS 420913,ISIS 420915, ISIS 420921, ISIS 420922, ISIS 420950, ISIS 420955, ISIS420957, or ISIS 420959.

The nine compounds having a nucleobase sequence of a sequence recited inSEQ ID NO: 25, 78, 80, 86, 87, 115, 120, 122 and 124, were furthertested for dose dependent inhibition in primary hepatocytes ofcynomolgus monkey (See Example 21). These compounds were also tested foroptimal viscosity (Example 22).The half life in the liver of CD1 mice ofseven of the compounds having a nucleobase sequence of a sequencerecited in SEQ ID NOs: 78, 86, 87. 115, 120 and 124 was also evaluated(Example 23).

Final evaluation of these studies (Examples 1-23), led to the selectionof eight compounds having a nucleobase sequence of a sequence recited inSEQ ID NO: 25, 80, 86, 87, 115, 120, 122 and 124. By virtue of theircomplementary sequence, the compounds are complementary to the regions504-523, 505-524, 512-531, 513-532, 577-596, 582-601, 584-603, and586-605 of SEQ ID NO: 1. In certain embodiments, the compounds targetingthe listed regions, as further described herein, comprise a modifiedoligonucleotide having some nucleobase portion of the sequence recitedin the SEQ ID NOs, as further described herein. In certain embodiments,the compounds targeting the listed regions or having a nucleobaseportion of a sequence recited in the listed SEQ ID NOs can be of variouslength, as further described herein, and can have one of various motifs,as further described herein. In certain embodiments, a compoundtargeting a region or having a nucleobase portion of a sequence recitedin the listed SEQ ID NOs has the specific length and motif as indicatedby the ISIS NOs: ISIS 304299, ISIS 420915, ISIS 420921, ISIS 420922,ISIS 420950, ISIS 420955, ISIS 420957, or ISIS 420959.

These eight compounds were tested for efficacy, pharmacokinetic profileand tolerability in cynomolgus monkeys (Example 24). The inhibitionstudies in these monkeys indicated that treatment with some of thesecompounds caused high inhibition of TTR mRNA in the liver. Specifically,treatment with ISIS 420950, ISIS 420955 and ISIS 420915 caused 91%, 79%and 78% inhibition, respectively compared to the PBS control. It wasnoted that ISIS 420915 caused greater inhibition of TTR (78%) mRNAcompared to ISIS 304299 (59%), even though the two oligonucleotidesdiffer from each other by a single base-pair shift of their targetregion on SEQ ID NO: 1. Protein analysis also complemented the RNAanalysis data with treatment with ISIS 420915 causing 76% inhibition andtreatment with ISIS 304299 causing 47% inhibition of TTR proteincompared to the control. RBP4 protein levels, as a protein associatedwith transthyretin, was also expected to decrease after treatment withthe antisense compounds. RBP4 protein levels decreased by 63% aftertreatment with ISIS 420915. Treatment with ISIS 304299 decreased RBP4protein levels by 19%. Additionally, ISIS 420915 was more tolerable thanISIS 304299, as indicated in the monkey study (Example 24) Transaminaselevels of monkeys treated with ISIS 304299 (ALT 81 IU/L and AST 101IU/L) were higher than those treated with ISIS 420915 (ALT 68 IU/L andAST 62 IU/L). The complement C3 levels of monkeys treated with ISIS304299 (96 mg/dL) were lower than that of monkeys treated with ISIS420915 (104 mg/dL).

Accordingly, provided herein are antisense compounds with any one ormore of the improved characteristics. In a certain embodiments, providedherein are compounds comprising a modified oligonucleotide as furtherdescribed herein targeted to or specifically hybridizable with theregion of nucleotides of SEQ ID NO: 1.

Accordingly, provided herein are antisense compounds with any one ormore of the improved characteristics. In a certain embodiments, providedherein are compounds comprising a modified oligonucleotide as furtherdescribed herein targeted to or specifically hybridizable with theregion of nucleotides of SEQ ID NO: 2.

Accordingly, provided herein are antisense compounds with any one ormore of the improved characteristics. In a certain embodiments, providedherein are compounds comprising a modified oligonucleotide as furtherdescribed herein targeted to or specifically hybridizable with theregion of nucleotides of SEQ ID NO: 4.

In certain embodiments, the compounds as described herein areefficacious by virtue of having at least one of an in vitro IC₅₀ of lessthan 2.9 uM, less than 2.2 uM, less than 2.0 uM, less than 1.5 uM, lessthan 1.4 uM, less than 1.3 uM, less than 1.0 uM, less than 0.7 uM, lessthan 0.6 uM, when delivered to a cynomolgous monkey hepatocyte cell lineusing electroporation as described in Example 67. In certainembodiments, the compounds as described herein are highly tolerable asdemonstrated by having at least one of an increase in ALT or AST valueof no more than 4 fold, 3 fold, or 2 fold over saline treated animals;or an increase in liver, spleen or kidney weight of no more than 30%,20%, 15%, 12%, 10%, 5% or 2%.

Certain Indications

In certain embodiments, provided herein are methods of treating anindividual comprising administering one or more pharmaceuticalcompositions as described herein. In certain embodiments, the individualhas central nervous system related disease.

As shown in the examples below, compounds targeted to transthyretin asdescribed herein have been shown to reduce the severity of physiologicalsymptoms of central nervous system related diseases. In certain of theexperiments, the compounds reduced rate of amyloid plaque formation,e.g., the animals continued to experience symptoms, but the symptomswere less severe compared to untreated animals. In other of theexperiments, however, the compounds appear to result in regeneration offunction over time; e.g., animals treated for a longer period of timeexperienced less severe symptoms than those administered the compoundsfor a shorter period of time. The ability of the compounds exemplifiedbelow to restore function therefore demonstrates that symptoms of thedisease may be reversed by treatment with a compound as describedherein.

Accordingly, provided herein are methods for ameliorating a symptomassociated with central nervous system related, cardiac, neuropathologicor gastrointestinal disease in a subject in need thereof. In certainembodiments, provided is a method for reducing the rate of onset of asymptom associated with central nervous system related, cardiac,neuropathologic or gastrointestinal disease. In certain embodiments,provided is a method for reducing the severity of a symptom associatedwith central nervous system related, cardiac, neuropathologic orgastrointestinal. In such embodiments, the methods compriseadministering to an individual in need thereof a therapeuticallyeffective amount of a compound targeted to a Transthyretin nucleic acid.

Transthyretin amyloidosis is characterized by numerous physical,neurological, psychiatric, and/or peripheral symptoms. Any symptom knownto one of skill in the art to be associated with transthyretinamyloidosis can be ameliorated or otherwise modulated as set forth abovein the methods described above. In certain embodiments, the symptom is aphysical, cognitive, psychiatric, or peripheral symptom. In certainembodiments, the symptom is a physical symptom selected from the groupconsisting of restlessness, lack of coordination, nystagmus, spasticparaparesis, lack of muscle coordination, impaired vision, insomnia,unusual sensations, myoclonus, blindness, loss of speech, Carpal tunnelsyndrome, seizures, subarachnoid hemorrhages, stroke and bleeding in thebrain, hydrocephalus, ataxia, and spastic paralysis, coma, sensoryneuropathy, parathesia, hypesthesia, motor neuropathy, autonomicneuropathy, orthostatic hypotension, cyclic constipation, cyclicdiarrhea, nausea, vomiting, reduced sweating, impotence, delayed gastricemptying, urinary retention, urinary incontinence, progressivecardiopathy, fatigue, shortness of breath, weight loss, lack ofappetite, numbness, tingling, weakness, enlarged tongue, nephroticsyndrome, congestive heart failure, dyspnea on exertion, peripheraledema, arrhythmias, palpitations, light-headedness, syncope, posturalhypotension, peripheral nerve problems, sensory motor impairment, lowerlimb neuropathy, upper limb neuropathy, hyperalgesia, alteredtemperature sensation, lower extremity weakness, cachexia, peripheraledema, hepatomegaly, purpura, diastolic dysfunction, prematureventricular contractions, cranial neuropathy, diminished deep tendonreflexes, amyloid deposits in the corpus vitreum, vitreous opacity, dryeyes, glaucoma, scalloped appearance in the pupils, swelling of the feetdue to water retention. In certain embodiments, the symptom is acognitive symptom selected from the group consisting of impaired memory,impaired judgment, and thinking, impaired planning, impairedflexibility, impaired abstract thinking, impaired rule acquisition,impaired initiation of appropriate actions, impaired inhibition ofinappropriate actions, impaired short-term memory, impaired long-termmemory, paranoia, disorientation, confusion, hallucination and dementia.In certain embodiments, the symptom is a psychiatric symptom selectedfrom the group consisting of dementia; anxiety, depression, bluntedaffect, egocentrisms, aggression, compulsive behavior, irritability,personality changes, including, impaired memory, judgment, and thinkingand suicidal ideation.

In certain embodiments, the symptom is restlessness. In certainembodiments, the symptom is lack of coordination. In certainembodiments, the symptom is nystagmus. In certain embodiments, thesymptom is spastic paraparesis. In certain embodiments, the symptom islack of muscle coordination. In certain embodiments, the symptom isimpaired vision. In certain embodiments, the symptom is insomnia. Incertain embodiments, the symptom is unusual sensations. In certainembodiments, the symptom is myoclonus. In certain embodiments, thesymptom is blindness. In certain embodiments, the symptom is loss ofspeech. In certain embodiments, the symptom is Carpal tunnel syndrome.In certain embodiments, the symptom is seizures. In certain embodiments,the symptom is subarachnoid hemorrhages. In certain embodiments, thesymptom is stroke. In certain embodiments, the symptom is bleeding inthe brain. In certain embodiments, the symptom is hydrocephalus. Incertain embodiments, the symptom is ataxia. In certain embodiments, thesymptom is spastic paralysis. In certain embodiments, the symptom iscoma. In certain embodiments, the symptom is sensory neuropathy. Incertain embodiments, the symptom is parathesia. In certain embodiments,the symptom is hypesthesia. In certain embodiments, the symptom is motorneuropathy. In certain embodiments, the symptom is autonomic neuropathy.In certain embodiments, the symptom is orthostatic hypotension. Incertain embodiments, the symptom is cyclic constipation. In certainembodiments, the symptom is cyclic diarrhea. In certain embodiments, thesymptom is nausea. In certain embodiments, the symptom is vomiting. Incertain embodiments, the symptom is reduced sweating. In certainembodiments, the symptom is impotence. In certain embodiments, thesymptom is delayed gastric emptying. In certain embodiments, the symptomis urinary retention. In certain embodiments, the symptom is urinaryincontinence. In certain embodiments, the symptom is progressivecardiopathy. In certain embodiments, the symptom is fatigue. In certainembodiments, the symptom is shortness of breath. In certain embodiments,the symptom is weight loss. In certain embodiments, the symptom isnumbness. In certain embodiments, the symptom is tingling. In certainembodiments, the symptom is weakness. In certain embodiments, thesymptom is enlarged tongue. In certain embodiments, the symptom isnephrotic syndrome. In certain embodiments, the symptom is congestiveheart failure. In certain embodiments, the symptom is dyspnea onexertion. In certain embodiments, the symptom is peripheral edema. Incertain embodiments, the symptom is arrhythmias. In certain embodiments,the symptom is palpitations. In certain embodiments, the symptom islight-headedness. In certain embodiments, the symptom is syncope. Incertain embodiments, the symptom is postural hypotension. In certainembodiments, the symptom is peripheral nerve problems. In certainembodiments, the symptom is sensory motor impairment. In certainembodiments, the symptom is lower limb neuropathy. In certainembodiments, the symptom is upper limb neuropathy. In certainembodiments, the symptom is hyperalgesia. In certain embodiments, thesymptom is altered temperature sensation. In certain embodiments, thesymptom is lower extremity weakness. In certain embodiments, the symptomis cachexia. In certain embodiments, the symptom is edema. In certainembodiments, the symptom is hepatomegaly. In certain embodiments, thesymptom is purpura. In certain embodiments, the symptom is diastolicdysfunction. In certain embodiments, the symptom is prematureventricular contractions. In certain embodiments, the symptom is cranialneuropathy. In certain embodiments, the symptom is diminished deeptendon reflexes. In certain embodiments, the symptom is amyloid depositsin the corpus vitreum. In certain embodiments, the symptom is vitreousopacity. In certain embodiments, the symptom is dry eyes. In certainembodiments, the symptom is glaucoma. In certain embodiments, thesymptom is scalloped appearance in the pupils. In certain embodiments,the symptom is swelling of the feet due to water retention.

In certain embodiments, the symptom is impaired memory. In certainembodiments, the symptom is impaired judgment, and thinking In certainembodiments, the symptom is impaired planning In certain embodiments,the symptom is impaired flexibility. In certain embodiments, the symptomis impaired abstract thinking. In certain embodiments, the symptom isimpaired rule acquisition. In certain embodiments, the symptom isimpaired initiation of appropriate actions. In certain embodiments, thesymptom is impaired inhibition of inappropriate actions. In certainembodiments, the symptom is impaired short-term memory. In certainembodiments, the symptom is impaired long-term memory. In certainembodiments, the symptom is paranoia. In certain embodiments, thesymptom is disorientation. In certain embodiments, the symptom isconfusion. In certain embodiments, the symptom is hallucination. Incertain embodiments, the symptom is dementia.

In certain embodiments, the symptom is dementia. In certain embodiments,the symptom is anxiety. In certain embodiments, the symptom isdepression. In certain embodiments, the symptom is blunted affect. Incertain embodiments, the symptom is egocentrisms. In certainembodiments, the symptom is aggression. In certain embodiments, thesymptom is compulsive behavior. In certain embodiments, the symptom isirritability. In certain embodiments, the symptom is personalitychanges. In certain embodiments, the symptom is suicidal ideation.

In certain embodiments, provided are methods of treating an individualcomprising administering one or more pharmaceutical compositions asdescribed herein. In certain embodiments, the individual has centralnervous system related disease.

In certain embodiments, administration of an antisense compound targetedto a transthyretin nucleic acid results in reduction of transthyretinexpression by at least about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 95 or 99%, or a range defined by any two of thesevalues.

In certain embodiments, pharmaceutical compositions comprising anantisense compound targeted to transthyretin are used for thepreparation of a medicament for treating a patient suffering orsusceptible to central nervous system related disease.

In certain embodiments, the methods described herein includeadministering a compound comprising a modified oligonucleotide having acontiguous nucleobases portion as described herein of a sequence recitedin SEQ ID NO: 25, 78, 80, 86, 87, 115, 120, 122 and 124.

Administration

In certain embodiments, the compounds and compositions as describedherein may be administered in a number of ways depending upon whetherlocal or systemic treatment is desired and upon the area to be treated.Administration may be topical, pulmonary, e.g., by inhalation orinsufflation of powders or aerosols, including by nebulizer;intratracheal, intranasal, epidermal and transdermal, oral orparenteral. The compounds and compositions as described herein can bedelivered in a manner to target a particular tissue, such as the liveror brain.

In certain embodiments, the compounds and compositions as describedherein are administered parenterally. “Parenteral administration” meansadministration through injection or infusion. Parenteral administrationincludes subcutaneous administration, intravenous administration,intramuscular administration, intraarterial administration,intraperitoneal administration, or intracranial administration, e.g.intracerebral administration, intrathecal administration,intraventricular administration, ventricular administration,intracerebroventricular administration, cerebral intraventricularadministration or cerebral ventricular administration. Administrationcan be continuous, or chronic, or short or intermittent.

In certain embodiments, parenteral administration is by infusion.Infusion can be chronic or continuous or short or intermittent. Incertain embodiments, infused pharmaceutical agents are delivered with apump. In certain embodiments, parenteral administration is by injection.

In certain embodiments, parenteral administration is subcutaneous.

In further embodiments, the formulation for administration is thecompounds described herein and saline.

In certain embodiments, compounds and compositions are delivered to theCNS. In certain embodiments, compounds and compositions are delivered tothe cerebrospinal fluid. In certain embodiments, compounds andcompositions are administered to the brain parenchyma. In certainembodiments, compounds and compositions are delivered to an animal intomultiple regions of the central nervous system (e.g., into multipleregions of the brain, and/or into the spinal cord) by intrathecaladministration, or intracerebroventricular administration. Broaddistribution of compounds and compositions, described herein, within thecentral nervous system may be achieved with intraparenchymaladministration, intrathecal administration, or intracerebroventricularadministration.

In certain embodiments, the present invention includes pharmaceuticalcompositions that can be delivered by injection directly into the brain.The injection can be by stereotactic injection into a particular regionof the brain (e.g., the substantia nigra, choroid plexus, cortex,hippocampus, striatum, choroid plexus or globus pallidus). The compoundcan also be delivered into diffuse regions of the brain (e.g., diffusedelivery to the cortex of the brain).

In certain embodiments, parenteral administration is by injection. Theinjection may be delivered with a syringe or a pump. In certainembodiments, the injection is a bolus injection. In certain embodiments,the injection is administered directly to a tissue, such as striatum,caudate, cortex, hippocampus and cerebellum.

In certain embodiments, delivery of a compound or composition describedherein can affect the pharmacokinetic profile of the compound orcomposition. In certain embodiments, injection of a compound orcomposition described herein, to a targeted tissue improves thepharmacokinetic profile of the compound or composition as compared toinfusion of the compound or composition. In a certain embodiment, theinjection of a compound or composition improves potency compared tobroad diffusion, requiring less of the compound or composition toachieve similar pharmacology. In certain embodiments, similarpharmacology refers to the amount of time that a target mRNA and/ortarget protein is down-regulated (e.g. duration of action). In certainembodiments, methods of specifically localizing a pharmaceutical agent,such as by bolus injection, decreases median effective concentration(EC50) by a factor of about 50 (e.g. 50 fold less concentration intissue is required to achieve the same or similar pharmacodynamiceffect). In certain embodiments, methods of specifically localizing apharmaceutical agent, such as by bolus injection, decreases medianeffective concentration (EC50) by a factor of 20, 25, 30, 35, 40, 45 or50. In certain embodiments the pharmaceutical agent in an antisensecompound as further described herein. In certain embodiments, thetargeted tissue is brain tissue. In certain embodiments the targetedtissue is striatal tissue. In certain embodiments, decreasing EC50 isdesirable because it reduces the dose required to achieve apharmacological result in a patient in need thereof

The half-life of MOE gapmer oligonucleotides in CD1 mice liver tissue isabout 21 days (see Examples 12).

In certain embodiments, an antisense oligonucleotide is delivered byinjection or infusion once every month, every two months, every 90 days,every 3 months, every 6 months, twice a year or once a year.

Certain Combination Therapies

In certain embodiments, one or more pharmaceutical compositions of thepresent invention are co-administered with one or more otherpharmaceutical agents. In certain embodiments, such one or more otherpharmaceutical agents are designed to treat the same disease, disorder,or condition as the one or more pharmaceutical compositions describedherein. In certain embodiments, such one or more other pharmaceuticalagents are designed to treat a different disease, disorder, or conditionas the one or more pharmaceutical compositions described herein. Incertain embodiments, such one or more other pharmaceutical agents aredesigned to treat an undesired side effect of one or more pharmaceuticalcompositions as described herein. In certain embodiments, one or morepharmaceutical compositions are co-administered with anotherpharmaceutical agent to treat an undesired effect of that otherpharmaceutical agent. In certain embodiments, one or more pharmaceuticalcompositions are co-administered with another pharmaceutical agent toproduce a combinational effect. In certain embodiments, one or morepharmaceutical compositions are co-administered with anotherpharmaceutical agent to produce a synergistic effect.

In certain embodiments, one or more pharmaceutical compositions and oneor more other pharmaceutical agents are administered at the same time.In certain embodiments, one or more pharmaceutical compositions and oneor more other pharmaceutical agents are administered at different times.In certain embodiments, one or more pharmaceutical compositions and oneor more other pharmaceutical agents are prepared together in a singleformulation. In certain embodiments, one or more pharmaceuticalcompositions and one or more other pharmaceutical agents are preparedseparately.

In certain embodiments, the second compound is administered prior toadministration of a pharmaceutical composition of the present invention.In certain embodiments, the second compound is administered followingadministration of a pharmaceutical composition of the present invention.In certain embodiments, the second compound is administered at the sametime as a pharmaceutical composition of the present invention. Incertain embodiments, the dose of a co-administered second compound isthe same as the dose that would be administered if the second compoundwas administered alone. In certain embodiments, the dose of aco-administered second compound is lower than the dose that would beadministered if the second compound was administered alone. In certainembodiments, the dose of a co-administered second compound is greaterthan the dose that would be administered if the second compound wasadministered alone.

In certain embodiments, the co-administration of a second compoundenhances the effect of a first compound, such that co-administration ofthe compounds results in an effect that is greater than the effect ofadministering the first compound alone. In certain embodiments, theco-administration results in effects that are additive of the effects ofthe compounds when administered alone. In certain embodiments, theco-administration results in effects that are supra-additive of theeffects of the compounds when administered alone. In certainembodiments, the first compound is an antisense compound. In certainembodiments, the second compound is an antisense compound.

In certain embodiments, pharmaceutical agents that may beco-administered with a pharmaceutical composition of the presentinvention include antipsychotic agents, such as, e.g., haloperidol,chlorpromazine, clozapine, quetapine, and olanzapine; antidepressantagents, such as, e.g., fluoxetine, sertraline hydrochloride, venlafaxineand nortriptyline; tranquilizing agents such as, e.g., benzodiazepines,clonazepam, paroxetine, venlafaxin, and beta-blockers; mood-stabilizingagents such as, e.g., lithium, valproate, lamotrigine, andcarbamazepine; paralytic agents such as, e.g., Botulinum toxin; and/orother experimental agents including, but not limited to, tetrabenazine(Xenazine), creatine, conezyme Q10, trehalose, docosahexanoic acids,ACR16, ethyl-EPA, atomoxetine, citalopram, dimebon, memantine, sodiumphenylbutyrate, ramelteon, ursodiol, zyprexa, xenasine, tiapride,riluzole, amantadine, [123I]MNI-420, atomoxetine, tetrabenazine,digoxin, detromethorphan, warfarin, alprozam, ketoconazole, omeprazole,and minocycline.

In certain embodiments, pharmaceutical agents that may beco-administered with a pharmaceutical composition of the presentinvention include analgesics, such as, paracetamol (acetaminophen);non-steroidal anti-inflammatory drugs (NSAIDs), such as, salicylates;narcotic drugs, such as, morphine, and synthetic drugs with narcoticproperties such as tramadol.

In certain embodiments, pharmaceutical agents that may beco-administered with a pharmaceutical composition of the presentinvention include muscle relaxants, such as, benzodiapines andmethocarbamol.

Formulations

The compounds of the invention may also be admixed, conjugated orotherwise associated with other molecules, molecule structures ormixtures of compounds, as for example, liposomes, receptor-targetedmolecules, or other formulations, for assisting in uptake, distributionand/or absorption. Representative United States patents that teach thepreparation of such uptake, distribution and/or absorption-assistingformulations include, but are not limited to, U.S. Pat. Nos.: 5,108,921;5,354,844; 5,416,016; 5,459,127; 5,521,291; 5,543,158; 5,547,932;5,583,020; 5,591,721; 4,426,330; 4,534,899; 5,013,556; 5,108,921;5,213,804; 5,227,170; 5,264,221; 5,356,633; 5,395,619; 5,416,016;5,417,978; 5,462,854; 5,469,854; 5,512,295; 5,527,528; 5,534,259;5,543,152; 5,556,948; 5,580,575; and 5,595,756, each of which is hereinincorporated by reference.

The antisense compounds of the invention encompass any pharmaceuticallyacceptable salts, esters, or salts of such esters, or any other compoundwhich, upon administration to an animal, including a human, is capableof providing (directly or indirectly) the biologically active metaboliteor residue thereof.

The term “pharmaceutically acceptable salts” refers to physiologicallyand pharmaceutically acceptable salts of the compounds of the invention:i.e., salts that retain the desired biological activity of the parentcompound and do not impart undesired toxicological effects thereto. Foroligonucleotides, preferred examples of pharmaceutically acceptablesalts and their uses are further described in U.S. Pat. No. 6,287,860,which is incorporated herein in its entirety. Sodium salts have beenshown to be suitable forms of oligonucleotide drugs.

The present invention also includes pharmaceutical compositions andformulations which include the antisense compounds of the invention. Thepharmaceutical compositions of the present invention may be administeredin a number of ways depending upon whether local or systemic treatmentis desired and upon the area to be treated. Administration may beparenteral. Parenteral administration includes intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; or intracranial, e.g., intracerebral administration,intrathecal administration, intraventricular administration, ventricularadministration, intracerebroventricular administration, cerebralintraventricular administration or cerebral ventricular administration.

Administration intraventricularly, is preferred to target transthyretinexpression in the choroid plexus. Oligonucleotides with at least one2′-O-methoxyethyl modification are believed to be particularly usefulfor oral administration. Pharmaceutical compositions and formulationsfor topical administration may include transdermal patches, ointments,lotions, creams, gels, drops, suppositories, sprays, liquids andpowders. Conventional pharmaceutical carriers, aqueous, powder or oilybases, thickeners and the like may be necessary or desirable. Coatedcondoms, gloves and the like may also be useful.

The pharmaceutical formulations of the present invention, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention may be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels, suppositories, andenemas. The compositions of the present invention may also be formulatedas suspensions in aqueous, non-aqueous or mixed media. Aqueoussuspensions may further contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, foams and liposome-containingformulations. The pharmaceutical compositions and formulations of thepresent invention may comprise one or more penetration enhancers,carriers, excipients or other active or inactive ingredients.

Emulsions are typically heterogenous systems of one liquid dispersed inanother in the form of droplets usually exceeding 0.1 μm in diameter.Emulsions may contain additional components in addition to the dispersedphases, and the active drug which may be present as a solution in theaqueous phase, oily phase or itself as a separate phase. Microemulsionsare included as an embodiment of the present invention. Emulsions andtheir uses are well known in the art and are further described in U.S.Pat. No. 6,287,860, which is incorporated herein in its entirety.

Formulations of the present invention include liposomal formulations. Asused in the present invention, the term “liposome” means a vesiclecomposed of amphiphilic lipids arranged in a spherical bilayer orbilayers. Liposomes are unilamellar or multilamellar vesicles which havea membrane formed from a lipophilic material and an aqueous interiorthat contains the composition to be delivered. Cationic liposomes arepositively charged liposomes which are believed to interact withnegatively charged DNA molecules to form a stable complex. Liposomesthat are pH-sensitive or negatively-charged are believed to entrap DNArather than complex with it. Both cationic and noncationic liposomeshave been used to deliver DNA to cells.

Liposomes also include “sterically stabilized” liposomes, a term which,as used herein, refers to liposomes comprising one or more specializedlipids that, when incorporated into liposomes, result in enhancedcirculation lifetimes relative to liposomes lacking such specializedlipids. Liposomes and their uses are further described in U.S. Pat. No.6,287,860, which is incorporated herein in its entirety.

In another embodiment of the invention, formulations of the presentinvention include saline formulations. In certain embodiment of theinvention, a formulation consists of the compounds described herein andsaline. In certain embodiments, a formulation consists essentially ofthe compounds described herein and saline. In certain embodiments, thesaline is pharmaceutically acceptable grade saline. In certainembodiments, the saline is buffered saline. In certain embodiments, thesaline is phosphate buffered saline (PBS).

In certain embodiments, a formulation excludes liposomes. In certainembodiments, the formulation excludes sterically stabilized liposomes.In certain embodiments, a formulation excludes phospholipids. In certainembodiments, the formulation consists essentially of the compoundsdescribed herein and saline and excludes liposomes.

The pharmaceutical formulations and compositions of the presentinvention may also include surfactants. Surfactants and their uses arefurther described in U.S. Pat. No. 6,287,860, which is incorporatedherein in its entirety.

In one embodiment, the present invention employs various penetrationenhancers to affect the efficient delivery of nucleic acids,particularly oligonucleotides. Penetration enhancers and their uses arefurther described in U.S. Pat. No. 6,287,860, which is incorporatedherein in its entirety.

One of skill in the art will recognize that formulations are routinelydesigned according to their intended use, i.e. route of administration.

Preferred formulations for topical administration include those in whichthe oligonucleotides of the invention are in admixture with a topicaldelivery agent such as lipids, liposomes, fatty acids, fatty acidesters, steroids, chelating agents and surfactants. Preferred lipids andliposomes include neutral (e.g. dioleoylphosphatidyl DOPE ethanolamine,dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline)negative (e.g. dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g.dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidylethanolamine DOTMA).

Compositions and formulations for parenteral administration, includingintravenous, intraarterial, subcutaneous, intraperitoneal, intramuscularinjection or infusion, or intracranial may include sterile aqueoussolutions which may also contain buffers, diluents and other suitableadditives such as, but not limited to, penetration enhancers, carriercompounds and other pharmaceutically acceptable carriers or excipients.

Certain embodiments of the invention provide pharmaceutical compositionscontaining one or more oligomeric compounds and one or more otherchemotherapeutic agents which function by a non-antisense mechanism.Examples of such chemotherapeutic agents include but are not limited tocancer chemotherapeutic drugs such as daunorubicin, daunomycin,dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin,bleomycin, mafosfamide, ifosfamide, cytosine arabinoside,bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D,mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen,dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine,mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea,nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine,6-thioguanine, cytarabine, 5-azacytidine, hydroxyurea, deoxycoformycin,4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU),5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol,vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan,topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol(DES). When used with the compounds of the invention, suchchemotherapeutic agents may be used individually (e.g., 5-FU andoligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for aperiod of time followed by MTX and oligonucleotide), or in combinationwith one or more other such chemotherapeutic agents (e.g., 5-FU, MTX andoligonucleotide, or 5-FU, radiotherapy and oligonucleotide).Anti-inflammatory drugs, including but not limited to nonsteroidalanti-inflammatory drugs and corticosteroids, and antiviral drugs,including but not limited to ribivirin, vidarabine, acyclovir andganciclovir, may also be combined in compositions of the invention.Combinations of antisense compounds and other non-antisense drugs arealso within the scope of this invention. Two or more combined compoundsmay be used together or sequentially.

In another related embodiment, compositions of the invention may containone or more antisense compounds, particularly oligonucleotides, targetedto a first nucleic acid and one or more additional antisense compoundstargeted to a second nucleic acid target. Alternatively, compositions ofthe invention may contain two or more antisense compounds targeted todifferent regions of the same nucleic acid target. Numerous examples ofantisense compounds are known in the art. Two or more combined compoundsmay be used together or sequentially.

Dosing

The formulation of therapeutic compositions and their subsequentadministration (dosing) is believed to be within the skill of those inthe art. Dosing is dependent on severity and responsiveness of thedisease state to be treated, with the course of treatment lasting fromseveral days to several months, or until a cure is effected or adiminution of the disease state is achieved. Optimal dosing schedulescan be calculated from measurements of drug accumulation in the body ofthe patient. Optimum dosages may vary depending on the relative potencyof individual oligonucleotides, and can generally be estimated based onEC50s found to be effective in in vitro and in vivo animal models. Ingeneral, dosage is from 0.01 μg to 100 g per kg of body weight, and maybe given once or more daily, weekly, monthly or yearly, or at desiredintervals. Following successful treatment, it may be desirable to havethe patient undergo maintenance therapy to prevent the recurrence of thedisease state, wherein the oligonucleotide is administered inmaintenance doses, ranging from 0.01 μg to 100 g per kg of body weight,once or more daily.

While the present invention has been described with specificity inaccordance with certain of its preferred embodiments, the followingexamples serve only to illustrate the invention and are not intended tolimit the same. Each of the references, GenBank accession numbers, andthe like recited in the present application is incorporated herein byreference in its entirety.

EXAMPLES Non-Limiting Disclosure and Incorporation by Reference

While certain compounds, compositions and methods described herein havebeen described with specificity in accordance with certain embodiments,the following examples serve only to illustrate the compounds describedherein and are not intended to limit the same. Each of the referencesrecited in the present application is incorporated herein by referencein its entirety.

Example 1 Antisense Inhibition of Human Transthyretin in HepG2 Cells

Antisense oligonucleotides were designed targeting a transthyretinnucleic acid and were tested for their effects on transthyretin mRNA invitro. Cultured HepG2 cells at a density of 10,000 cells per well weretransfected using lipofectin reagent with 50 nM antisenseoligonucleotide. After a treatment period of approximately 24 hours, RNAwas isolated from the cells and transthyretin mRNA levels were measuredby quantitative real-time PCR. Human primer probe set RTS1396 (forwardsequence CCCTGCTGAGCCCCTACTC, designated herein as SEQ ID NO: 5; reversesequence TCCCTCATTCCTTGGGATTG, designated herein as SEQ ID NO: 6; probesequence ATTCCACCACGGCTGTCGTCAX , designated herein as SEQ ID NO: 7).Transthyretin mRNA levels were adjusted according to total RNA content,as measured by RIBOGREEN®. Results are presented as percent inhibitionof transthyretin, relative to untreated control cells.

The chimeric antisense oligonucleotides in Tables 1 and 2 were designedas 5-10-5 MOE gapmers. The gapmers are 20 nucleotides in length, whereinthe central gap segment is comprised of ten 2′-deoxynucleotides and isflanked on both sides (in the 5′ and 3′ directions) by wings comprisingfive nucleotides each. Each nucleotide in the 5′ wing segment and eachnucleotide in the 3′ wing segment has a 2′-MOE modification. Theinternucleoside linkages throughout each gapmer are phosphorothioate(P═S) linkages. All cytidine residues throughout each gapmer are5-methylcytidines. “Human Target start site” indicates the 5′-mostnucleotide to which the gapmer is targeted in the human gene sequence.“Human Target stop site” indicates the 3′-most nucleotide to which thegapmer is targeted human gene sequence. Each gapmer listed in Table 1 istargeted to human transthyretin mRNA, designated herein as SEQ ID NO: 1(GENBANK Accession No. NM_000371.2). Certain gapmers were also designedwhich targeted intronic sequences or intron-exon junctions of the humantransthyretin genomic sequence, designated herein as SEQ ID NO: 2(GENBANK Accession No. NT_010966.10 truncated from nucleotides 2009236to 2017289) and are listed in Table 2.

The human oligonucleotides of Tables 1 and 2 are also cross-reactivewith rhesus monkey gene sequences. ‘Mismatches’ indicate the number ofnucleobases by which the human oligonucleotide is mismatched with arhesus monkey gene sequence. The greater the complementarity between thehuman oligonucleotide and the rhesus monkey sequence, the more likelythe human oligonucleotide can cross-react with the rhesus monkeysequence. The human oligonucleotides in Table 1 were compared to exons1-4 extracted from the rhesus monkey genomic sequence GENBANK AccessionNo. NW_001105671.1, based on similarity to human exons. The humanoligonucleotides in Table 2 were compared to the rhesus monkey genomicsequence, designated herein as SEQ ID NO: 4 (GENBANK Accession No.NW_001105671.1 truncated from nucleotides 628000 to 638000). “Rhesusmonkey Target start site” indicates the 5′-most nucleotide to which thegapmer is targeted in the rhesus monkey gene sequence. “Rhesus monkeyTarget stop site” indicates the 3′-most nucleotide to which the gapmeris targeted rhesus monkey gene sequence.

TABLE 1 Inhibition of human transthyretin mRNA levels by chimeric antisense oligonucleotides having 5-10-5 MOE wings and  deoxy gap targeted to SEQ ID NO: 1 and SEQ ID NO: 4 Rhesus RhesusHuman Human monkey monkey SEQ ISIS Start Stop % start stop Mis- ID NOSite Site Region Sequence inhibition site site matches NO 304267 217 236coding ACTGGTTTTCC 53 217 236 0   8 CAGAGGCAA 304268 222 241 codingGACTCACTGGT 16 222 241 0   9 TTTCCCAGA 304280 353 372 coding TGAATACCAC51 353 372 0  10 CTCTGCATGC 304284 425 444 coding CCGTGGTGGA 82 425 4440  11 ATAGGAGTAG 304285 427 446 coding AGCCGTGGTG 89 427 446 0  12GAATAGGAGT 304286 431 450 coding CGACAGCCGT 63 431 450 0  13 GGTGGAATAG304287 438 457 coding TTGGTGACGA 88 438 457 0  14 CAGCCGTGGT 304288 440459 coding GATTGGTGAC 82 440 459 0  15 GACAGCCGTG 304289 442 461 codingGGGATTGGTG 78 442 461 0  16 ACGACAGCCG 304290 443 462 coding TGGGATTGGT85 443 462 0  17 GACGACAGCC 304291 449 468 coding- ATTCCTTGGGA 52 449468 0  18 stop TTGGTGACG codon 304292 450 469 coding- CATTCCTTGGG 34 450469 0  19 stop ATTGGTGAC codon 304293 451 470 coding- TCATTCCTTGG 29 451470 0  20 stop GATTGGTGA codon 304294 460 479 coding- AGAAGTCCCT 32 460479 0  21 stop CATTCCTTGG codon- 3′UTR 304296 481 500 3′- GTCCTTCAGGT 84478 497 2  22 UTR CCACTGGAG 304297 489 508 3′- CATCCCTCGTC  0 486 505 1 23 UTR CTTCAGGTC 304298 501 520 3′- TACATGAAAT 26 498 517 0  24 UTRCCCATCCCTC 304299 507 526 3′- CTTGGTTACAT 85 504 523 0  25 UTR GAAATCCCA304300 513 532 3′- AATACTCTTGG 49 510 529 0  26 UTR TTACATGAA 304301 526545 3′UTR TTAGTAAAAA  0 523 542 0  27 TGGAATACTC 304302 532 551 3′UTRACTGCTTTAGT 42 529 548 0  28 AAAAATGGA 304303 539 558 3′UTR TGAAAACACT41 536 555 0  29 GCTTTAGTAA 304304 546 565 3′UTR TATGAGGTGA 49 543 562 0 30 AAACACTGCT 304307 564 583 3′UTR TGGACTTCTAA 73 561 580 2  31CATAGCATA 304308 572 591 3′UTR TCTCTGCCTGG 55 569 588 1  32 ACTTCTAAC304309 578 597 3′- TTATTGTCTCT 77 575 594 0  33 UTR GCCTGGACT 304311 597616 3′- TGCCTTTCACA 80 594 613 0  34 UTR GGAATGTTT 304312 598 617 3′-GTGCCTTTCAC 71 595 614 0  35 UTR AGGAATGTT 420871  36  55 codingCAGAGGAGGA 48  36  55 0  36 GCAGACGATG 420872 120 139 coding TCTAGAACTTT55 120 139 0  37 GACCATCAG 420873 212 231 coding TTTTCCCAGAG 54 212 2310  38 GCAAATGGC 420874 226 245 coding TCCAGACTCAC 63 226 245 0  39TGGTTTTCC 420875 271 290 coding TATCCCTTCTA 40 271 290 0  40 CAAATTCCT420876 285 304 coding ATTTCCACTTT 42 285 304 0  41 GTATATCCC 420877 293312 coding TGGTGTCTATT 76 293 312 0  42 TCCACTTTG 420878 303 322 codingCAGTAAGATTT 80 303 322 0  43 GGTGTCTAT 420879 307 326 coding CTTCCAGTAAG73 307 326 0  44 ATTTGGTGT 420880 347 366 coding CCACCTCTGCA 76 347 3660  45 TGCTCATGG 420881 355 374 coding TGTGAATACC 58 355 374 0  46ACCTCTGCAT 420882 357 376 coding GCTGTGAATA 69 357 376 0  47 CCACCTCTGC420883 362 381 coding CGTTGGCTGTG 64 362 381 0  48 AATACCACC 420884 428447 coding CAGCCGTGGT 93 428 447 0  49 GGAATAGGAG 420885 430 449 codingGACAGCCGTG 93 430 449 0  50 GTGGAATAGG 420886 432 451 coding ACGACAGCCG92 432 451 0  51 TGGTGGAATA 420887 433 452 coding GACGACAGCC 93 433 4520  52 GTGGTGGAAT 420888 434 453 coding TGACGACAGC 95 434 453 0  53CGTGGTGGAA 420889 435 454 coding GTGACGACAG 93 435 454 0  54 CCGTGGTGGA420890 436 455 coding GGTGACGACA 97 436 455 0  55 GCCGTGGTGG 420891 437456 coding TGGTGACGAC 97 437 456 0  56 AGCCGTGGTG 420892 439 458 codingATTGGTGACG 93 439 458 0  57 ACAGCCGTGG 420893 441 460 coding GGATTGGTGA96 441 460 0  58 CGACAGCCGT 420894 444 463 coding TTGGGATTGGT 88 444 4630  59 GACGACAGC 420895 445 464 coding CTTGGGATTGG 95 445 464 0  60TGACGACAG 420896 446 465 coding CCTTGGGATTG 95 446 465 0  61 GTGACGACA420897 447 466 coding TCCTTGGGATT 94 447 466 0  62 GGTGACGAC 420898 448467 coding- TTCCTTGGGAT 86 448 467 0  63 stop TGGTGACGA codon 420899 452471 coding- CTCATTCCTTG 94 452 471 0  64 stop GGATTGGTG codon- 3′UTR420900 453 472 coding- CCTCATTCCTT 92 453 472 0  65 stop GGGATTGGTcodon- 3′UTR 420901 454 473 coding- CCCTCATTCCT 93 454 473 0  66 stopTGGGATTGG codon- 3′UTR 420902 455 474 coding- TCCCTCATTCC 75 455 474 0 67 stop TTGGGATTG codon- 3′UTR 420903 456 475 coding- GTCCCTCATTC 57456 475 0  68 stop CTTGGGATT codon- 3′UTR 420904 457 476 coding-AGTCCCTCATT 62 457 476 0  69 stop CCTTGGGAT codon- 3′UTR 420905 458 477coding- AAGTCCCTCAT 58 458 477 0  70 stop TCCTTGGGA codon- 3′UTR 420906459 478 coding- GAAGTCCCTC 79 459 478 0  71 stop ATTCCTTGGG codon- 3′UTR420907 461 480 coding- GAGAAGTCCC 59 461 480 0  72 stop TCATTCCTTGcodon- 3′UTR 420908 462 481 coding- GGAGAAGTCC 75 462 481 0  73 stopCTCATTCCTT codon- 3′UTR 420909 500 519 3′UTR ACATGAAATC 82 497 516 0  74CCATCCCTCG 420910 502 521 3′UTR TTACATGAAAT 74 499 518 0  75 CCCATCCCT420911 503 522 3′UTR GTTACATGAA 81 500 519 0  76 ATCCCATCCC 420912 504523 3′UTR GGTTACATGA 92 501 520 0  77 AATCCCATCC 420913 505 524 3′UTRTGGTTACATGA 95 502 521 0  78 AATCCCATC 420914 506 525 3′UTR TTGGTTACATG93 503 522 0  79 AAATCCCAT 420915 508 527 3′UTR TCTTGGTTACA 92 505 524 0 80 TGAAATCCC 420916 509 528 3′UTR CTCTTGGTTAC 88 506 525 0  81ATGAAATCC 420917 510 529 3′UTR ACTCTTGGTTA 92 507 526 0  82 CATGAAATC420918 511 530 3′UTR TACTCTTGGTT 88 508 527 0  83 ACATGAAAT 420919 512531 3′UTR ATACTCTTGGT 89 509 528 0  84 TACATGAAA 420920 514 533 3′UTRGAATACTCTTG 87 511 530 0  85 GTTACATGA 420921 515 534 3′UTR GGAATACTCTT92 512 531 0  86 GGTTACATG 420922 516 535 3′UTR TGGAATACTCT 95 513 532 0 87 TGGTTACAT 420923 517 536 3′UTR ATGGAATACT 90 514 533 0  88CTTGGTTACA 420924 518 537 3′UTR AATGGAATAC 75 515 534 0  89 TCTTGGTTAC420925 519 538 3′UTR AAATGGAATA 87 516 535 0  90 CTCTTGGTTA 420926 520539 3′UTR AAAATGGAAT 88 517 536 0  91 ACTCTTGGTT 420927 521 540 3′UTRAAAAATGGAA 50 518 537 0  92 TACTCTTGGT 420928 522 541 3′UTR TAAAAATGGA26 519 538 0  93 ATACTCTTGG 420929 523 542 3′UTR GTAAAAATGG 56 520 539 0 94 AATACTCTTG 420930 524 543 3′UTR AGTAAAAATG 18 521 540 0  95GAATACTCTT 420931 525 544 3′UTR TAGTAAAAAT 12 522 541 0  96 GGAATACTCT420932 527 546 3′UTR TTTAGTAAAA  1 524 543 0  97 ATGGAATACT 420933 528547 3′UTR CTTTAGTAAAA  0 525 544 0  98 ATGGAATAC 420934 529 548 3′UTRGCTTTAGTAAA  6 526 545 0  99 AATGGAATA 420935 530 549 3′UTR TGCTTTAGTAA 0 527 546 0 100 AAATGGAAT 420936 531 550 3′UTR CTGCTTTAGTA 40 528 547 0101 AAAATGGAA 420937 533 552 3′UTR CACTGCTTTAG 47 530 549 0 102TAAAAATGG 420938 534 553 3′UTR ACACTGCTTTA 30 531 550 0 103 GTAAAAATG420939 535 554 3′UTR AACACTGCTTT  0 532 551 0 104 AGTAAAAAT 420940 536555 3′UTR AAACACTGCTT  0 533 552 0 105 TAGTAAAAA 420941 537 556 3′UTRAAAACACTGC  0 534 553 0 106 TTTAGTAAAA 420942 538 557 3′UTR GAAAACACTG 0 535 554 0 107 CTTTAGTAAA 420943 540 559 3′UTR GTGAAAACAC 14 537 556 0108 TGCTTTAGTA 420944 541 560 3′UTR GGTGAAAACA 43 538 557 0 109CTGCTTTAGT 420945 542 561 3′UTR AGGTGAAAAC 41 539 558 0 110 ACTGCTTTAG420946 543 562 3′UTR GAGGTGAAAA 20 540 559 0 111 CACTGCTTTA 420947 544563 3′UTR TGAGGTGAAA 69 541 560 0 112 ACACTGCTTT 420948 545 564 3′UTRATGAGGTGAA 63 542 561 0 113 AACACTGCTT 420949 579 598 3′UTR TTTATTGTCTC84 576 595 0 114 TGCCTGGAC 420950 580 599 3′UTR TTTTATTGTCT 69 577 596 0115 CTGCCTGGA 420951 581 600 3′UTR GTTTTATTGTC 87 578 597 0 116TCTGCCTGG 420952 582 601 3′UTR TGTTTTATTGT 67 579 598 0 117 CTCTGCCTG420953 583 602 3′UTR ATGTTTTATTG 51 580 599 0 118 TCTCTGCCT 420954 584603 3′UTR AATGTTTTATT 60 581 600 0 119 GTCTCTGCC 420955 585 604 3′UTRGAATGTTTTAT 65 582 601 0 120 TGTCTCTGC 420956 586 605 3′UTR GGAATGTTTTA67 583 602 0 121 TTGTCTCTG 420957 587 606 3′UTR AGGAATGTTTT 68 584 603 0122 ATTGTCTCT 420958 588 607 3′UTR CAGGAATGTTT 45 585 604 0 123TATTGTCTC 420959 589 608 3′UTR ACAGGAATGT 28 586 605 0 124 TTTATTGTCT

TABLE 2Inhibition of human transthyretin mRNA levels by chimeric antisenseoligonucleotides having 5-10-5 MOE wings and deoxy gap targeted toSEQ ID NO: 2 and SEQ ID NO: 4 Rhesus Rhesus Human Human monkey monkeySEQ ISIS Start Stop % start stop Mis- ID NO Site Site Region Sequenceinhibition site site matches NO 420960  606  625 exon1- GATGTCACAG 131755 1774 0 125 intron1 AAACACTCAC 420961  665  684 intron GCAAAGCTGG  71814 1833 0 126 1 AAGGAGTCAC 420962  748  767 intron GAACTTCATTC  0 18971916 0 127 1 TTTTTGAAG 420963  882  901 intron AGCTTCCTTAA  0 2031 20500 128 1 TATCATATC 420964  966  985 intron TATAGGGCCA 10 2115 2134 0 1291 GAATATAATC 420965 1010 1029 intron ACTAAGCCTTT 17 2159 2178 0 130 1TAAAGATTA 420966 1208 1227 intron TGGAATTACT 35 2356 2375 0 131 1GAAAAGATGT 420967 1289 1308 intron ACCAGGGATG 43 2437 2456 0 132 1TGTATAATGA 420968 1364 1383 intron TCCCTACTCAG  0 2512 2531 0 133 1TATAACACA 420969 1472 1491 intron GATCAGAGTG  0 2620 2639 0 134 1AAAGGATTTA 420970 1687 1706 intron GGGAAGATAA 46 2826 2845 0 135 2AACCAAGTCC 420971 1739 1758 intron TAAATTCTTTA  0 2878 2897 0 136 2GCAGATGAT 420972 1842 1861 intron AATGATGCTC 23 2980 2999 0 137 2AGGTTCCTGG 420973 2051 2070 intron TTGGTGTTACC  0 3187 3206 0 138 2CAGGGACAC 420974 2207 2226 intron AAAGTGTTCA 29 3344 3363 0 139 2TTAGGCAAAA 420975 2655 2674 intron GGCATTTTATA  0 3798 3817 0 140 2TAAACATAA 420976 2733 2752 intron AAGAACATTG  0 3876 3895 0 141 2GAATATTTTT 420977 2874 2893 intron GTTGGAAATT  9 4017 4036 0 142 2GCTTCCCATT 420978 3015 3034 intron AGTGGAAAAC  0 4156 4175 0 143 2CTAAAGTAGG 420979 3618 3637 intron TTCCCCTCAAC  0 4795 4814 0 144 2TAAGTCAGA 420980 3735 3754 intron2- CCTATAAGGT  0 4930 4949 0 145 exon 3GTGAAAGTCT 420981 4096 4115 intron TGTAAGTTCA  0 5291 5310 0 146 3AGTCATGTTA 420982 4306 4325 intron GTGTTGCCAA  0 5502 5521 0 147 3GAATCACTTG 420983 4404 4423 intron AAAACACTTA  0 5600 5619 0 148 3TAATTGTGTC 420984 4518 4537 intron CTTTGACAAGT  0 5714 5733 0 149 3TATTTGACT 420985 4880 4899 intron ATCCATGACT  0 6073 6092 0 150 3AAGCCAGAGA 420986 5185 5204 intron ATGGTTCCCAT  0 6379 6398 0 151 3CAGGCTGAG 420987 5542 5561 intron GCATTTATCAG  0 6732 6751 0 152 3AAGAAGCTG 420988 6030 6049 intron TTGACCTTCAG  0 7226 7245 0 153 3CCCACTTGA 420989 6133 6152 intron AGGAAGTGAG  0 7641 7660 0 154 3AATCACCTAA 420990 6320 6339 intron AGAAGACAGT  0 7828 7847 0 155 3AAAGATGTGT 420991 6457 6476 intron AAATTGTGGA  0 7966 7985 0 156 3TCAAAATGCT 420992 6736 6755 intron AACCAGACTT  0 8246 8265 0 157 3GAATTATTGT 420993 6811 6830 intron AGTGGCTGCC  0 8321 8340 0 158 3AACCACAGAC 420994 7106 7125 intron GGAAGTCCAG  0 8615 8634 0 159 3TGCCAACTTA 420995 7162 7181 intron ATCCATTTCCA  0 8670 8689 0 160 3CCAGAGCCC

Due to the short length of the human transthyretin mRNA, a second primerprobe set was designed away from the first primer probe set, RTS1396, toavoid amplicon oligonucleotides. The antisense oligonucleotides werealso tested for their effects on transthyretin mRNA in vitro using newhuman primer probe set RTS3029 (forward sequenceCTTGCTGGACTGGTATTTGTGTCT, designated herein as SEQ ID NO: 161, reversesequence AGAACTTTGACCATCAGAGGACACT, designated herein as SEQ ID NO: 162;probe sequence CCCTACGGGCACCGGTGAATCCX, designated herein as SEQ ID NO:163). Cultured HepG2 cells at a density of 10,000 cells per well weretransfected using lipofectin reagent with 50 nM antisenseoligonucleotide. After a treatment period of approximately 24 hours, RNAwas isolated from the cells and transthyretin mRNA levels were measuredby quantitative real-time PCR. Transthyretin mRNA levels were adjustedaccording to total RNA content, as measured by RIBOGREEN®. Results arepresented as percent inhibition of transthyretin, relative to untreatedcontrol cells. The results are presented in Table 3 as percentinhibition of the PBS control cell set.

TABLE 3 Inhibition of human transthyretin mRNA levels by chimericantisense oligonucleotides having 5-10-5 MOE wings and deoxy gap withprimer probe set RTS3029 ISIS NO Region % inhibition 304267 coding 13304268 coding 10 304280 coding 23 304284 coding 10 304285 coding 34304286 coding 0 304287 coding 34 304288 coding 45 304289 coding 3 304290coding 16 304291 coding-stop codon 4 304292 coding-stop codon 10 304293coding-stop codon 14 304294 stop codon-3′ UTR 30 304296 exon 4 78 304297exon 4 29 304298 exon 4 19 304299 exon 4 85 304300 exon 4 52 304301 exon4 15 304302 exon 4 45 304303 exon 4 51 304304 exon 4 62 304307 exon 4 76304308 exon 4 63 304309 exon 4 75 304311 exon 4 81 304312 exon 4 68420871 coding 0 420872 coding 5 420873 coding 19 420874 coding 0 420875coding 6 420876 coding 20 420877 coding 28 420878 coding 37 420879coding 34 420880 coding 36 420881 coding 10 420882 coding 27 420883coding 13 420884 coding 28 420885 coding 4 420886 coding 21 420887coding 39 420888 coding 37 420889 coding 9 420890 coding 27 420891coding 39 420892 coding 43 420893 coding 39 420894 coding 0 420895coding 0 420896 coding 24 420897 coding 31 420898 coding- 0 420899 stopcodon-3′UTR 41 420900 stop codon-3′UTR 26 420901 stop codon-3′UTR 28420902 stop codon-3′UTR 20 420903 stop codon-3′UTR 20 420904 stopcodon-3′UTR 22 420905 stop codon-3′UTR 32 420906 stop codon-3′UTR 13420907 -stop codon-3′UTR 0 420908 stop codon-3′UTR 45 420909 3′UTR 41420910 3′UTR 14 420911 3′UTR 45 420912 3′UTR 62 420913 3′UTR 81 4209143′UTR 68 420915 3′UTR 71 420916 3′UTR 54 420917 3′UTR 50 420918 3′UTR 43420919 3′UTR 65 420920 3′UTR 61 420921 3′UTR 65 420922 3′UTR 68 4209233′UTR 62 420924 3′UTR 9 420925 3′UTR 17 420926 3′UTR 47 420927 3′UTR 57420928 3′UTR 51 420929 3′UTR 46 420930 3′UTR 39 420931 3′UTR 14 4209323′UTR 6 420933 3′UTR 1 420934 3′UTR 48 420935 3′UTR 13 420936 3′UTR 62420937 3′UTR 65 420938 3′UTR 48 420939 3′UTR 7 420940 3′UTR 3 4209413′UTR 31 420942 3′UTR 0 420943 3′UTR 40 420944 3′UTR 78 420945 3′UTR 58420946 3′UTR 52 420947 3′UTR 71 420948 3′UTR 73 420949 3′UTR 88 4209503′UTR 82 420951 3′UTR 90 420952 3′UTR 82 420953 3′UTR 71 420954 3′UTR 67420955 3′UTR 73 420956 3′UTR 65 420957 3′UTR 74 420958 3′UTR 69 4209593′UTR 63 420960 exon1-intron1 14 420961 intron 1 16 420962 intron 1 0420963 intron 1 0 420964 intron 1 14 420965 intron 1 23 420966 intron 125 420967 intron 1 12 420968 intron 1 0 420969 intron 1 0 420970 intron2 25 420971 intron 2 0 420972 intron 2 25 420973 intron 2 7 420974intron 2 28 420975 intron 2 9 420976 intron 2 21 420977 intron 2 14420978 intron 2 37 420979 intron 2 37 420980 intron2-exon 3 16 420981intron 3 0 420982 intron 3 28 420983 intron 3 0 420984 intron 3 0 420985intron 3 0 420986 intron 3 7 420987 intron 3 0 420988 intron 3 0 420989intron 3 0 420990 intron 3 6 420991 intron 3 15 420992 intron 3 0 420993intron 3 0 420994 intron 3 0 420995 intron 3 10

Based on the inhibition results using the new primer probe set RTS3029,antisense oligonucleotides exhibiting 50% or more inhibition oftransthyretin mRNA were selected for further studies.

Example 2 Antisense Inhibition of Human Transthyretin in HepG2 Cells byOligonucleotides Designed by Microwalk

Additional gapmers were designed based on the gapmers presented in Table3 that demonstrated an inhibition of at least 50%. These gapmers weredesigned by creating gapmers shifted slightly upstream and downstream(i.e. “microwalk”) of the original gapmers from Table 3. Gapmers werealso created with various motifs, e.g. 5-10-5 MOE, 3-14-3 MOE, 2-13-5MOE, and 4-11-5 MOE motifs. These gapmers were tested in vitro. CulturedHepG2 cells at a density of 10,000 cells per well were transfected usinglipofectin reagent with 50 nM antisense oligonucleotide. After atreatment period of approximately 24 hours, RNA was isolated from thecells and transthyretin mRNA levels were measured by quantitativereal-time PCR. The human primer probe set RTS3029 was used to measuretransthyretin mRNA levels. Transthyretin mRNA levels were adjustedaccording to total RNA content, as measured by RIBOGREEN®. Results arepresented as percent inhibition of transthyretin, relative to untreatedcontrol cells. The results are presented in Table 4.

The chimeric antisense oligonucleotides in Table 4 were designed as5-10-5 MOE, 3-14-3 MOE, 2-13-5 MOE or 4-11-5 MOE gapmers. The gapmersdesignated with an asterisk (*) in Table 4 are the original gapmers fromwhich gapmers, ISIS 425650-425763, were designed via microwalk. The5-10-5 gapmers are 20 nucleotides in length, wherein the central gapsegment is comprised of ten 2′-deoxynucleotides and is flanked on bothsides (in the 5′ and 3′ directions) by wings comprising five nucleotideseach. The 3-14-3 gapmers are 20 nucleotides in length, wherein thecentral gap segment is comprised of fourteen 2′-deoxynucleotides and isflanked on both sides (in the 5′ and 3′ directions) by wings comprisingthree nucleotides each. The 2-13-5 gapmers are 20 nucleotides in length,wherein the central gap segment is comprised of thirteen2′-deoxynucleotides and is flanked on the 5′ and the 3′ directions withwings comprising two and five nucleotides respectively. The 4-11-5gapmers are 20 nucleotides in length, wherein the central gap segment iscomprised of eleven 2′-deoxynucleotides and is flanked on the 5′ and the3′ directions with wings comprising four and five nucleotidesrespectively. For each of the motifs (5-10-5, 3-14-3, 2-113-5, and4-11-5), each nucleotide in the 5′ wing segment and each nucleotide inthe 3′ wing segment has a 2′-MOE modification. The internucleosidelinkages throughout each gapmer are phosphorothioate (P═S) linkages. Allcytidine residues throughout each gapmer are 5-methylcytidines. “Targetstart site” indicates the 5′-most nucleotide to which the gapmer istargeted. “Target stop site” indicates the 3′-most nucleotide to whichthe gapmer is targeted. Each gapmer listed in Table 4 is targeted to thetarget region spanning nucleobases 481-619 of SEQ ID NO: 1 (GENBANKAccession No. NM_000371.2).

As shown in Table 4, several of the gapmers exhibited at least 50%inhibition, including ISIS numbers: 304296, 425655, 425695, 425735,425649, 425656, 425696, 425736, 420912, 425657, 425697, 425737, 420913,425658, 425698, 425738, 420914, 425659, 425699, 425739, 304299, 425660,425700, 425740, 420915, 420916, 425662, 425702, 420919, 425703, 420920,425664, 425704, 425742, 420921, 425665, 425705, 425743, 420922, 425666,425706, 420923, 420937, 420944, 425669, 425709, 425746, 425710, 425711,425747, 420948, 425712, 425748, 425673, 425713, 425749, 425651, 425675,425715, 425751, 304309, 425676, 425716, 425752, 420949, 425677, 425717,425753, 420950, 425678, 425718, 425754, 420951, 425679, 425719, 425755,420952, 425680, 425720, 425756, 420953, 425681, 425721, 425757, 420954,425722, 425758, 420955, 425759, 425724, 425760, 425762, 304310, 425729,425764, 425653, 425690, 425730, 425765, 304311, 425691, 425731, 425766,304312, 425692, 425732, 425767, 425654, 425693, 425733, 425768, 304313,425734, and 425769.

Several of the gapmers exhibited at least 60% inhibition, including ISISnumbers: 304296, 425655, 425695, 425735, 425649, 425656, 425696, 425736,420912, 425657, 425697, 425737, 420913, 425658, 425698, 425738, 420914,425659, 425739, 304299, 425740, 420915, 425702, 420919, 420920, 425742,420921, 425665, 425705, 425706, 420923, 425746, 425711, 425747, 420948,425712, 425748, 425651, 425715, 425751, 304309, 425716, 425752, 425677,425717, 425753, 420950, 425718, 425754, 420951, 425679, 425719, 425755,420952, 425680, 425720, 420953, 425681, 425721, 425757, 420954, 425722,425758, 420955, 425724, 425760, 425764, 425653, 425690, 425730, 425765,304311, 425691, 425731, 425766, 304312, 425692, 425732, 425767, 425654,425693, 425733, 304313, and 425769.

Several of the gapmers exhibited at least 70% inhibition, including ISISnumbers: 304296, 425655, 425695, 425735, 425649, 425656, 425696, 425736,420912, 425657, 425737, 420913, 425738, 420914, 425659, 304299, 420915,420920, 425742, 425712, 425748, 425716, 425754, 420951, 425679, 425719,425755, 425680, 425721, 425757, 425760, 425653, 425690, 425730, 425765,304311, 425691, 425731, 425766, 304312, 425767, 425693, and 304313.

Several of the gapmers exhibited at least 80% inhibition, including ISISnumbers: 304296, 425655, 425695, 425736, 420913, 425659, 304299, 420915,425716, 425754, 425719, 425757, 425765, and 425767.

Several of the gapmers exhibited at least 85% inhibition, including ISISnumbers: 420913, 425716, 425754, and 425719.

One gapmer, ISIS 425719, exhibited 90% inhibition.

TABLE 4 Inhibition of human transthyretin mRNA levels by  chimeric antisense oligonucleotides targeted to   SEQ ID NO: 1 (GENBANK Accession No. NM_000371.2) SEQ Start Stop % IDOligoID Site Site Sequence Motif inhibition NO *304296 481 500GTCCTTCAGGTCCACTGGAG 5-10-5 83  22  425655 481 500 GTCCTTCAGGTCCACTGGAG3-14-3 80  22  425695 481 500 GTCCTTCAGGTCCACTGGAG 2-13-5 80  22  425735481 500 GTCCTTCAGGTCCACTGGAG 4-11-5 72  22  425649 482 501CGTCCTTCAGGTCCACTGGA 5-10-5 75 170  425656 482 501 CGTCCTTCAGGTCCACTGGA3-14-3 78 170  425696 482 501 CGTCCTTCAGGTCCACTGGA 2-13-5 74 170  425736482 501 CGTCCTTCAGGTCCACTGGA 4-11-5 83 170 *420912 504 523GGTTACATGAAATCCCATCC 5-10-5 73  77  425657 504 523 GGTTACATGAAATCCCATCC3-14-3 76  77  425697 504 523 GGTTACATGAAATCCCATCC 2-13-5 69  77  425737504 523 GGTTACATGAAATCCCATCC 4-11-5 78  77 *420913 505 524TGGTTACATGAAATCCCATC 5-10-5 89  78  425658 505 524 TGGTTACATGAAATCCCATC3-14-3 69  78  425698 505 524 TGGTTACATGAAATCCCATC 2-13-5 61  78  425738505 524 TGGTTACATGAAATCCCATC 4-11-5 78  78 *420914 506 525TTGGTTACATGAAATCCCAT 5-10-5 70  79  425659 506 525 TTGGTTACATGAAATCCCAT3-14-3 83  79  425699 506 525 TTGGTTACATGAAATCCCAT 2-13-5 56  79  425739506 525 TTGGTTACATGAAATCCCAT 4-11-5 69  79 *304299 507 526CTTGGTTACATGAAATCCCA 5-10-5 83  25  425660 507 526 CTTGGTTACATGAAATCCCA3-14-3 59  25  425700 507 526 CTTGGTTACATGAAATCCCA 2-13-5 52  25  425740507 526 CTTGGTTACATGAAATCCCA 4-11-5 69  25 *420915 508 527TCTTGGTTACATGAAATCCC 5-10-5 81  80  425661 508 527 TCTTGGTTACATGAAATCCC3-14-3 48  80  425701 508 527 TCTTGGTTACATGAAATCCC 2-13-5 41  80  425741508 527 TCTTGGTTACATGAAATCCC 4-11-5 37  80 *420916 509 528CTCTTGGTTACATGAAATCC 5-10-5 52  81  425662 509 528 CTCTTGGTTACATGAAATCC3-14-3 57  81  425702 509 528 CTCTTGGTTACATGAAATCC 2-13-5 63  81 *420919512 531 ATACTCTTGGTTACATGAAA 5-10-5 69  84  425663 512 531ATACTCTTGGTTACATGAAA 3-14-3 46  84  425703 512 531 ATACTCTTGGTTACATGAAA2-13-5 52  84 *420920 514 533 GAATACTCTTGGTTACATGA 5-10-5 71  85  425664514 533 GAATACTCTTGGTTACATGA 3-14-3 57  85  425704 514 533GAATACTCTTGGTTACATGA 2-13-5 58  85  425742 514 533 GAATACTCTTGGTTACATGA4-11-5 71  85 *420921 515 534 GGAATACTCTTGGTTACATG 5-10-5 68  86  425665515 534 GGAATACTCTTGGTTACATG 3-14-3 65  86  425705 515 534GGAATACTCTTGGTTACATG 2-13-5 60  86  425743 515 534 GGAATACTCTTGGTTACATG4-11-5 56  86 *420922 516 535 TGGAATACTCTTGGTTACAT 5-10-5 54  87  425666516 535 TGGAATACTCTTGGTTACAT 3-14-3 56  87  425706 516 535TGGAATACTCTTGGTTACAT 2-13-5 64  87  425744 516 535 TGGAATACTCTTGGTTACAT4-11-5 39  87 *420923 517 536 ATGGAATACTCTTGGTTACA 5-10-5 62  88  425667517 536 ATGGAATACTCTTGGTTACA 3-14-3 44  88  425707 517 536ATGGAATACTCTTGGTTACA 2-13-5 30  88 *420937 533 552 CACTGCTTTAGTAAAAATGG5-10-5 59 102  425668 533 552 CACTGCTTTAGTAAAAATGG 3-14-3 37 102  425708533 552 CACTGCTTTAGTAAAAATGG 2-13-5 32 102  425745 533 552CACTGCTTTAGTAAAAATGG 4-11-5 43 102 *420944 541 560 GGTGAAAACACTGCTTTAGT5-10-5 52 109  425669 541 560 GGTGAAAACACTGCTTTAGT 3-14-3 54 109  425709541 560 GGTGAAAACACTGCTTTAGT 2-13-5 54 109  425746 541 560GGTGAAAACACTGCTTTAGT 4-11-5 60 109 *420945 542 561 AGGTGAAAACACTGCTTTAG5-10-5 38 110  425670 542 561 AGGTGAAAACACTGCTTTAG 3-14-3 38 110  425710542 561 AGGTGAAAACACTGCTTTAG 2-13-5 52 110 *420947 544 563TGAGGTGAAAACACTGCTTT 5-10-5 34 112  425671 544 563 TGAGGTGAAAACACTGCTTT3-14-3 27 112  425711 544 563 TGAGGTGAAAACACTGCTTT 2-13-5 68 112  425747544 563 TGAGGTGAAAACACTGCTTT 4-11-5 61 112 *420948 545 564ATGAGGTGAAAACACTGCTT 5-10-5 66 113  425672 545 564 ATGAGGTGAAAACACTGCTT3-14-3 47 113  425712 545 564 ATGAGGTGAAAACACTGCTT 2-13-5 70 113  425748545 564 ATGAGGTGAAAACACTGCTT 4-11-5 71 113 *304304 546 565TATGAGGTGAAAACACTGCT 5-10-5 46  30  425673 546 565 TATGAGGTGAAAACACTGCT3-14-3 51  30  425713 546 565 TATGAGGTGAAAACACTGCT 2-13-5 50  30  425749546 565 TATGAGGTGAAAACACTGCT 4-11-5 58  30  425650 547 566ATATGAGGTGAAAACACTGC 5-10-5 28 171  425674 547 566 ATATGAGGTGAAAACACTGC3-14-3 40 171  425714 547 566 ATATGAGGTGAAAACACTGC 2-13-5 44 171  425750547 566 ATATGAGGTGAAAACACTGC 4-11-5 47 171  425651 577 596TATTGTCTCTGCCTGGACTT 5-10-5 65 172  425675 577 596 TATTGTCTCTGCCTGGACTT3-14-3 55 172  425715 577 596 TATTGTCTCTGCCTGGACTT 2-13-5 65 172  425751577 596 TATTGTCTCTGCCTGGACTT 4-11-5 62 172 *304309 578 597TTATTGTCTCTGCCTGGACT 5-10-5 66  33  425676 578 597 TTATTGTCTCTGCCTGGACT3-14-3 59  33  425716 578 597 TTATTGTCTCTGCCTGGACT 2-13-5 87  33  425752578 597 TTATTGTCTCTGCCTGGACT 4-11-5 67  33 *420949 579 598TTTATTGTCTCTGCCTGGAC 5-10-5 57 114  425677 579 598 TTTATTGTCTCTGCCTGGAC3-14-3 67 114  425717 579 598 TTTATTGTCTCTGCCTGGAC 2-13-5 68 114  425753579 598 TTTATTGTCTCTGCCTGGAC 4-11-5 69 114 *420950 580 599TTTTATTGTCTCTGCCTGGA 5-10-5 61 115  425678 580 599 TTTTATTGTCTCTGCCTGGA3-14-3 59 115  425718 580 599 TTTTATTGTCTCTGCCTGGA 2-13-5 69 115  425754580 599 TTTTATTGTCTCTGCCTGGA 4-11-5 86 115 *420951 581 600GTTTTATTGTCTCTGCCTGG 5-10-5 78 116  425679 581 600 GTTTTATTGTCTCTGCCTGG3-14-3 73 116  425719 581 600 GTTTTATTGTCTCTGCCTGG 2-13-5 90 116  425755581 600 GTTTTATTGTCTCTGCCTGG 4-11-5 73 116 *420952 582 601TGTTTTATTGTCTCTGCCTG 5-10-5 61 117  425680 582 601 TGTTTTATTGTCTCTGCCTG3-14-3 77 117  425720 582 601 TGTTTTATTGTCTCTGCCTG 2-13-5 67 117  425756582 601 TGTTTTATTGTCTCTGCCTG 4-11-5 57 117 *420953 583 602ATGTTTTATTGTCTCTGCCT 5-10-5 65 118  425681 583 602 ATGTTTTATTGTCTCTGCCT3-14-3 61 118  425721 583 602 ATGTTTTATTGTCTCTGCCT 2-13-5 77 118  425757583 602 ATGTTTTATTGTCTCTGCCT 4-11-5 83 118 *420954 584 603AATGTTTTATTGTCTCTGCC 5-10-5 63 119  425682 584 603 AATGTTTTATTGTCTCTGCC3-14-3 42 119  425722 584 603 AATGTTTTATTGTCTCTGCC 2-13-5 69 119  425758584 603 AATGTTTTATTGTCTCTGCC 4-11-5 61 119 *420955 585 604GAATGTTTTATTGTCTCTGC 5-10-5 65 120  425683 585 604 GAATGTTTTATTGTCTCTGC3-14-3 30 120  425723 585 604 GAATGTTTTATTGTCTCTGC 2-13-5 44 120  425759585 604 GAATGTTTTATTGTCTCTGC 4-11-5 50 120 *420956 586 605GGAATGTTTTATTGTCTCTG 5-10-5 47 121  425684 586 605 GGAATGTTTTATTGTCTCTG3-14-3 44 121  425724 586 605 GGAATGTTTTATTGTCTCTG 2-13-5 65 121 *420957587 606 AGGAATGTTTTATTGTCTCT 5-10-5 37 122  425685 587 606AGGAATGTTTTATTGTCTCT 3-14-3 46 122  425725 587 606 AGGAATGTTTTATTGTCTCT2-13-5 43 122  425760 587 606 AGGAATGTTTTATTGTCTCT 4-11-5 78 122 *420958588 607 CAGGAATGTTTTATTGTCTC 5-10-5 41 123  425686 588 607CAGGAATGTTTTATTGTCTC 3-14-3  6 123  425726 588 607 CAGGAATGTTTTATTGTCTC2-13-5 41 123  425761 588 607 CAGGAATGTTTTATTGTCTC 4-11-5 39 123 *420959589 608 ACAGGAATGTTTTATTGTCT 5-10-5 43 124  425687 589 608ACAGGAATGTTTTATTGTCT 3-14-3 22 124  425727 589 608 ACAGGAATGTTTTATTGTCT2-13-5 25 124  425762 589 608 ACAGGAATGTTTTATTGTCT 4-11-5 57 124  425652590 609 CACAGGAATGTTTTATTGTC 5-10-5 23 173  425688 590 609CACAGGAATGTTTTATTGTC 3-14-3 11 173  425728 590 609 CACAGGAATGTTTTATTGTC2-13-5 37 173  425763 590 609 CACAGGAATGTTTTATTGTC 4-11-5 38 173  304310595 614 CCTTTCACAGGAATGTTTTA 5-10-5 57 174  425689 595 614CCTTTCACAGGAATGTTTTA 3-14-3 38 174  425729 595 614 CCTTTCACAGGAATGTTTTA2-13-5 58 174  425764 595 614 CCTTTCACAGGAATGTTTTA 4-11-5 60 174  425653596 615 GCCTTTCACAGGAATGTTTT 5-10-5 79 175  425690 596 615GCCTTTCACAGGAATGTTTT 3-14-3 73 175  425730 596 615 GCCTTTCACAGGAATGTTTT2-13-5 76 175  425765 596 615 GCCTTTCACAGGAATGTTTT 4-11-5 83 175 *304311597 616 TGCCTTTCACAGGAATGTTT 5-10-5 71  34  425691 597 616TGCCTTTCACAGGAATGTTT 3-14-3 74  34  425731 597 616 TGCCTTTCACAGGAATGTTT2-13-5 73  34  425766 597 616 TGCCTTTCACAGGAATGTTT 4-11-5 79  34 *304312598 617 GTGCCTTTCACAGGAATGTT 5-10-5 71  35  425692 598 617GTGCCTTTCACAGGAATGTT 3-14-3 69  35  425732 598 617 GTGCCTTTCACAGGAATGTT2-13-5 67  35  425767 598 617 GTGCCTTTCACAGGAATGTT 4-11-5 83  35  425654599 618 AGTGCCTTTCACAGGAATGT 5-10-5 64 176  425693 599 618AGTGCCTTTCACAGGAATGT 3-14-3 79 176  425733 599 618 AGTGCCTTTCACAGGAATGT2-13-5 68 176  425768 599 618 AGTGCCTTTCACAGGAATGT 4-11-5 50 176  304313600 619 AAGTGCCTTTCACAGGAATG 5-10-5 73 177  425694 600 619AAGTGCCTTTCACAGGAATG 3-14-3 45 177  425734 600 619 AAGTGCCTTTCACAGGAATG2-13-5 55 177  425769 600 619 AAGTGCCTTTCACAGGAATG 4-11-5 62 177

Example 3 Dose-Dependent Antisense Inhibition of Human Transthyretin inHepG2 Cells

Gapmers from Example 2 exhibiting significant in vitro inhibition ofhuman transthyretin were tested at various doses in HepG2 cells. Cellswere plated at a density of 20,000 cells per well and transfected usingelectroporation with 625 nM, 1250 nM, 2500 nM, 5000 nM and 10000 nMconcentrations of antisense oligonucleotide, as specified in Table 5.After a treatment period of approximately 16 hours, RNA was isolatedfrom the cells and transthyretin mRNA levels were measured byquantitative real-time PCR. Human transthyretin primer probe set RTS3029was used to measure mRNA levels. Transthyretin mRNA levels were adjustedaccording to total RNA content, as measured by RIBOGREEN®. Results arepresented as percent inhibition of transthyretin, relative to untreatedcontrol cells.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotideis also presented in Table 5 and was calculated by plotting theconcentrations of oligonucleotides used versus the percent inhibition oftransthyretin mRNA expression achieved at each concentration, and notingthe concentration of oligonucleotide at which 50% inhibition oftransthyretin mRNA expression was achieved compared to the control. Asillustrated in Table 5, transthyretin mRNA levels were significantlyreduced in a dose-dependent manner in antisense oligonucleotide treatedcells.

TABLE 5 Dose-dependent antisense inhibition of human transthyretin inHepG2 cells using electroporation ISIS 625 1250 2500 5000 10000 IC₅₀ NOnM nM nM nM nM (μM) 304296 57 74 83 91 96 <0.625 304299 43 76 82 95 940.627 420913 59 75 90 88 98 <0.625 420915 60 85 91 95 99 <0.625 42095164 77 90 97 99 <0.625 425653 70 86 86 88 82 <0.625 425655 48 80 85 97 96<0.625 425656 70 89 92 92 96 <0.625 425659 46 56 68 82 93 0.8 425679 6377 72 94 97 <0.625 425680 28 79 85 93 98 0.8 425693 2 64 74 76 81 1.7425695 74 87 91 97 98 <0.625 425716 69 84 95 97 98 <0.625 425719 58 8492 96 98 <0.625 425721 40 75 89 95 98 0.7 425736 64 71 86 93 93 <0.625425737 78 93 95 97 98 <0.625 425738 40 77 88 94 95 0.7 425754 56 75 8796 99 <0.625 425755 58 84 88 94 97 <0.625 425757 62 82 94 97 99 <0.625425760 58 42 74 85 93 <0.625 425765 81 86 87 83 88 <0.625 425766 83 8981 75 74 <0.625 425767 56 75 83 81 80 <0.625

Gapmers from Example 2 were also tested at various doses in HepG2 cellsusing the transfection reagent, lipofectin. Cells were plated at adensity of 10,000 cells per well and transfected using electroporationwith 6.25 nM, 12.5 nM, 25 nM, 50 nM and 100 nM concentrations ofantisense oligonucleotide, as specified in Table 6. After a treatmentperiod of approximately 16 hours, RNA was isolated from the cells andtransthyretin mRNA levels were measured by quantitative real-time PCR.Human transthyretin primer probe set RTS3029 was used to measure mRNAlevels. Transthyretin mRNA levels were adjusted according to total RNAcontent, as measured by RIBOGREEN®. Results are presented as percentinhibition of transthyretin, relative to untreated control cells. Asillustrated in Table 6, transthyretin mRNA levels were significantlyreduced in a dose-dependent manner in antisense oligonucleotide treatedcells.

TABLE 6 Dose-dependent antisense inhibition of human transthyretin inHepG2 cells using lipofectin reagent ISIS 6.25 12.5 25 50 100 IC₅₀ NO nMnM nM nM nM (nM) 304296 26 41 43 52 65 39 304299 22 70 43 74 83 20420913 4 60 60 68 82 30 420915 36 31 46 64 67 28 420951 10 37 56 85 8419 425653 25 38 60 74 77 18 425655 27 15 62 79 81 16 425656 37 62 47 6982 15 425659 17 35 33 79 73 30 425679 32 6 63 79 77 14 425680 16 48 4184 84 28 425693 10 19 51 66 61 26 425695 36 23 54 76 84 28 425716 57 5236 85 81 38 425719 25 39 28 60 76 45 425721 0 22 38 73 75 32 425736 2560 30 77 80 22 425737 36 52 50 60 76 14 425738 13 15 19 65 70 27 4257548 18 38 75 71 42 425755 26 46 54 77 86 20 425757 0 37 81 83 71 19 42576028 46 72 70 80 18 425765 0 52 48 66 69 29 425766 24 19 48 69 71 29425767 41 49 48 65 75 14

Example 4 Dose-Dependent Antisense Inhibition of Human Transthyretin inHepG2 Cells

Gapmers selected from Example 3 were tested at various doses in HepG2cells. Cells were plated at a density of 20,000 cells per well andtransfected using electroporation with 0.0617 μM, 0.1852 μM, 0.5556 μM,1.6667 μM and 5 μM concentrations of antisense oligonucleotide, asspecified in Table 7. After a treatment period of approximately 16hours, RNA was isolated from the cells and transthyretin mRNA levelswere measured by quantitative real-time PCR. Human transthyretin primerprobe set RTS3029 was used to measure mRNA levels. Transthyretin mRNAlevels were adjusted according to total RNA content, as measured byRIBOGREEN®. Results are presented as percent inhibition oftransthyretin, relative to untreated control cells. As illustrated inTable 7, transthyretin mRNA levels were reduced in a dose-dependentmanner in antisense oligonucleotide treated cells.

TABLE 7 Dose-dependent antisense inhibition of human transthyretin inHepG2 cells using electroporation ISIS 0.0617 0.1852 0.5556 1.6667 5IC₅₀ NO μM μM μM μM μM (μM) 304296 0 6 44 58 83 1.2 304299 38 10 57 8392 0.6 420913 51 51 54 73 93 0.2 420915 33 35 62 65 93 0.2 420951 40 3336 82 96 0.4 425653 55 58 74 72 84 <0.06 425655 8 35 54 57 90 0.5 42565612 43 43 78 94 0.4 425659 14 35 19 46 82 0.6 425679 30 13 23 69 91 0.8425680 0 35 45 74 84 0.7 425693 0 6 14 32 59 3.4 425695 15 47 61 81 910.3 425716 20 17 53 77 91 0.6 425719 0 14 45 78 94 0.8 425721 0 0 22 7484 0.9 425736 42 43 56 76 91 0.3 425737 21 29 61 81 97 0.3 425738 14 3957 74 93 0.4 425754 29 34 45 78 94 0.4 425755 8 21 57 78 95 0.5 42575729 28 62 83 95 0.4 425760 3 6 9 56 77 1.4 425765 24 51 75 77 88 0.3425766 7 41 59 73 77 0.3 425767 1 18 49 66 79 1.0

Example 5 Dose Response Confirmation of Antisense OligonucleotidesTargeting Human Transthyretin in Hep3B Cells

Gapmers from Example 4 exhibiting significant in vitro inhibition ofhuman transthyretin were tested at various doses in Hep3B cells. Cellswere plated at a density of 20,000 cells per well and transfected usingelectroporation with 0.0206 μM, 0.062 μM, 0.185 μM, 0.556 μM, 1.667 μMand 5 μM concentrations of antisense oligonucleotide, as specified inTable 8. After a treatment period of approximately 16 hours, RNA wasisolated from the cells and transthyretin mRNA levels were measured byquantitative real-time PCR. Human transthyretin primer probe set RTS1396was used to measure mRNA levels. Transthyretin mRNA levels were adjustedaccording to total RNA content, as measured by RIBOGREEN®. Results arepresented as percent inhibition of transthyretin, relative to untreatedcontrol cells. As illustrated in Table 8, transthyretin mRNA levels werereduced in a dose-dependent manner in antisense oligonucleotide treatedcells. The IC₅₀ of each oligonucleotide is also presented in Table 8.

TABLE 8 Dose-dependent antisense inhibition of human transthyretin inHep3B cells using electroporation ISIS 0.0206 0.062 0.185 0.556 1.667 5IC₅₀ NO μM μM μM μM μM μM (μM) 304299 27 2 25 52 76 96 0.5 420915 0 1227 30 69 93 0.8 425653 23 13 55 86 88 91 0.1 425655 3 30 32 62 84 94 0.3425656 0 0 29 66 82 95 0.5 425679 0 21 36 71 92 97 0.3 425695 37 23 6379 94 98 0.1 425736 31 43 40 64 82 95 0.1 425737 0 13 62 82 95 98 0.2425755 17 8 18 69 86 98 0.4 425757 22 47 53 79 96 98 0.2

Example 6 Dose Response Confirmation of Antisense OligonucleotidesTargeting Human Transthyretin in Human Transthyretin-Transgenic MousePrimary Hepatocytes

Gapmers from Example 5 were also tested at various doses in primaryhepatocytes of human transthyretin-transgenic mice. ISIS 304309, ISIS304311, ISIS 304312 and ISIS 420951 (see Example 2) were also retestedalong with these gapmers under the same culture conditions. Cells wereplated at a density of 10,000 cells per well and transfected usingcytofectin with 18.75 nM, 37.5 nM, 75 nM, 150 nM and 300 nMconcentrations of antisense oligonucleotide, as specified in Table 9.After a treatment period of approximately 16 hours, RNA was isolatedfrom the cells and transthyretin mRNA levels were measured byquantitative real-time PCR. Human transthyretin primer probe set RTS1396was used to measure mRNA levels. Transthyretin mRNA levels were adjustedaccording to total RNA content, as measured by RIBOGREEN®. Results arepresented as percent inhibition of transthyretin, relative to untreatedcontrol cells. As illustrated in Table 9, transthyretin mRNA levels werereduced in a dose-dependent manner in antisense oligonucleotide treatedcells.

TABLE 9 Dose-dependent antisense inhibition of human transthyretin inmouse primary hepatocytes using cytofectin ISIS 18.75 37.5 75 150 300 NOnM nM nM nM nM Motif 304299 54 79 97 98 99 5-10-5 304309 48 77 94 99 995-10-5 304311 45 79 92 96 98 5-10-5 304312 33 71 89 96 98 5-10-5 42091540 70 92 98 99 5-10-5 420951 41 86 96 98 99 5-10-5 425653 44 81 93 96 995-10-5 425655 61 88 96 99 99 3-14-3 425656 61 84 94 98 99 3-14-3 42567974 78 97 98 99 3-14-3 425695 66 84 96 98 99 2-13-5 425736 58 84 95 98 994-11-5 425737 57 77 95 98 99 4-11-5 425755 61 82 96 99 99 4-11-5 42575737 77 93 98 98 4-11-5

Example 7 Dose Response Confirmation of Antisense OligonucleotidesTargeting Human Transthyretin in HepG2 Cells

Gapmers from Example 6 were tested at various doses in HepG2 cells.Cells were plated at a density of 10,000 cells per well and transfectedusing electroporation with 0.062 μM, 0.185 μM, 0.556 μM, 1.66 μM and5000 μM concentrations of antisense oligonucleotide, as specified inTable 10. After a treatment period of approximately 16 hours, RNA wasisolated from the cells and transthyretin mRNA levels were measured byquantitative real-time PCR. Human transthyretin primer probe set RTS1396was used to measure mRNA levels. Transthyretin mRNA levels were adjustedaccording to total RNA content, as measured by RIBOGREEN®. Results arepresented as percent inhibition of transthyretin, relative to untreatedcontrol cells. As illustrated in Table 10, transthyretin mRNA levelswere reduced in a dose-dependent manner in antisense oligonucleotidetreated cells.

TABLE 10 Dose-dependent antisense inhibition of human transthyretin inHepG2 cells using electroporation ISIS 0.062 0.185 0.556 1.667 5.000IC₅₀ NO μM μM μM μM μM (μM) Motif 304299 55 66 72 87 96 0.037 5-10-5304309 41 65 72 91 96 0.087 5-10-5 304311 57 83 88 89 83 0.001 5-10-5304312 46 69 74 84 81 0.038 5-10-5 420915 38 62 80 90 98 0.096 5-10-5420951 45 71 84 93 97 0.049 5-10-5 425653 48 73 87 88 82 0.017 5-10-5425655 40 57 77 85 95 0.105 3-14-3 425656 28 54 70 94 97 0.177 3-14-3425679 43 51 81 95 99 0.106 3-14-3 425695 49 67 90 96 99 0.043 2-13-5425736 32 63 85 95 98 0.108 4-11-5 425737 42 71 90 98 99 0.053 4-11-5425755 24 63 85 95 99 0.137 4-11-5 425757 21 62 86 96 99 0.148 4-11-5

Example 8 Dose Response Confirmation of Antisense OligonucleotidesTargeting Human Transthyretin in Human Transthyretin-Transgenic MousePrimary Hepatocytes

Gapmers from Example 6 were also tested at various doses in primaryhepatocytes of human transthyretin-transgenic mice. Cells were plated ata density of 10,000 cells per well and transfected using cytofectin with5 nM, 10 nM, 20 nM, 40 nM and 80 nM concentrations of antisenseoligonucleotide, as specified in Table 11. After a treatment period ofapproximately 16 hours, RNA was isolated from the cells andtransthyretin mRNA levels were measured by quantitative real-time PCR.Human transthyretin primer probe set RTS3029 was used to measure mRNAlevels. Transthyretin mRNA levels were adjusted according to total RNAcontent, as measured by RIBOGREEN®. Results are presented as percentinhibition of transthyretin, relative to untreated control cells. Asillustrated in Table 11, transthyretin mRNA levels were reduced in adose-dependent manner in antisense oligonucleotide treated cells.

TABLE 11 Dose-dependent antisense inhibition of human transthyretin inmouse primary hepatocytes using cytofectin ISIS 5 10 20 40 80 NO nM nMnM nM nM Motif 304299 0 8 37 69 90 5-10-5 304309 0 9 39 75 93 5-10-5304311 1 13 43 70 81 5-10-5 304312 0 3 32 64 76 5-10-5 420915 0 0 34 5987 5-10-5 420951 0 12 57 84 92 5-10-5 425653 0 9 44 72 84 5-10-5 4256550 19 45 80 91 3-14-3 425656 0 2 33 70 93 3-14-3 425679 0 13 42 72 903-14-3 425695 3 12 33 70 90 2-13-5 425736 2 7 37 70 89 4-11-5 425737 0 436 65 89 4-11-5 425755 0 25 50 75 94 4-11-5 425757 0 5 43 72 92 4-11-5

Gapmers were also tested using electroporation as the transfectionagent. Cells were plated at a density of 35,000 cells per well andtransfected using electroporation with 148.148 nM, 444.444 nM, 1,333.333nM, 4,000 nM and 12,000 nM concentrations of antisense oligonucleotide,as specified in Table 12. After a treatment period of approximately 16hours, RNA was isolated from the cells and transthyretin mRNA levelswere measured by quantitative real-time PCR. Human transthyretin primerprobe set RTS3029 was used to measure mRNA levels. Transthyretin mRNAlevels were adjusted according to total RNA content, as measured byRIBOGREEN®. Results are presented as percent inhibition oftransthyretin, relative to untreated control cells.

TABLE 12 Dose-dependent antisense inhibition of human transthyretin inmouse primary hepatocytes using electroporation ISIS 148.148 444.4441333.333 4000 12000 NO nM nM nM nM nM Motif 304299 75 96 98 98 99 5-10-5304309 72 96 98 98 98 5-10-5 304311 68 92 93 94 97 5-10-5 304312 50 8492 93 97 5-10-5 420915 55 89 96 96 97 5-10-5 420951 65 92 95 96 985-10-5 425653 68 89 91 93 95 5-10-5 425655 63 94 96 96 96 3-14-3 42565669 93 98 98 98 3-14-3 425679 63 92 97 98 98 3-14-3 425695 69 92 96 96 952-13-5 425736 75 93 96 96 96 4-11-5 425737 71 94 96 96 95 4-11-5 42575570 93 95 95 95 4-11-5 425757 61 91 95 95 95 4-11-5

Example 9 Dose Response Confirmation of Antisense OligonucleotidesTargeting Human Transthyretin in Cynomolgus Monkey Primary Hepatocytes

Gapmers from Example 6 were also tested at various doses in primaryhepatocytes of cynomolgus monkeys. Cells were plated at a density of35,000 cells per well and transfected using electroporation with 1,250nM, 2,500 nM, 5,000 nM, 10,000 nM and 20,000 nM concentrations ofantisense oligonucleotide, as specified in Table 13. After a treatmentperiod of approximately 16 hours, RNA was isolated from the cells andtransthyretin mRNA levels were measured by quantitative real-time PCR.Human transthyretin primer probe set RTS1396 was used to measure mRNAlevels. Transthyretin mRNA levels were adjusted according to total RNAcontent, as measured by RIBOGREEN®. Results are presented as percentinhibition of transthyretin, relative to untreated control cells. Asillustrated in Table 13, transthyretin mRNA levels were reduced in adose-dependent manner in hepatocytes treated with ISIS oligonucleotides.

In absence of a complete cynomolgus monkey gene sequence in the NCBIdatabase, the oligonucleotides were tested for cross-reactivity againstthe rhesus monkey gene sequence, since the two species are from the samegenus, ‘Macaca’. The human oligonucleotides are cross-reactive withrhesus monkey transthyretin gene, designated herein as SEQ ID NO: 4(exons 1-4 extracted from GENBANK Accession No. NW_001105671.1).‘Mismatches’ indicates the number of mismatches between the humanoligonucleotide and the rhesus monkey transthyretin gene. ‘n/a’indicates that the human oligonucleotide has more than 3 mismatches withthe rhesus monkey transthyretin gene and therefore does not cross-reactwith it.

TABLE 13 Dose-dependent antisense inhibition of human transthyretin inRhesus monkey primary hepatocytes using electroporation Rhesus Rhesusmonkey monkey ISIS 1,250 2,500 5,000 10,000 2,0000 IC₅₀ Target startTarget stop Mis- NO nM nM nM nM nM (μM) site site matches 304299 21 4569 80 95 3.1 504 523 0 304309 53 66 79 85 93 <1.25 575 594 0 304311 7578 82 86 90 <1.25 594 613 0 304312 37 53 65 75 80 2.3 595 614 0 42091559 54 77 87 94 <1.25 505 524 0 420951 67 77 91 93 96 <1.25 578 597 0425653 56 72 84 83 85 <1.25 593 612 0 425655 0 7 0 21 45 >20 478 497 2425656 41 20 38 53 51 8.7 479 498 2 425679 68 74 88 94 98 <1.25 578 5970 425695 42 29 41 49 65 25.8 478 497 2 425736 36 27 37 49 74 8.2 479 4982 425737 76 78 89 95 97 <1.25 501 520 0 425755 79 80 92 94 97 <1.25 578597 0 425757 68 74 88 95 96 <1.25 580 599 0

Example 10 In Vivo Inhibition of Human Transthyretin in HumanTransthyretin-Transgenic Mice

Gapmers from Example 6, demonstrating significant inhibition oftransthyretin mRNA, were tested in transgenic mice containing the humantransthyretin gene and the efficacy of the gapmers was evaluated.

Treatment

Fifteen groups of four hTTR transgenic female mice each wereadministered subcutaneously twice a week for four weeks with 25 mg/kg ofISIS 304299, ISIS 304309, ISIS 304311, ISIS 304312, ISIS 420915, ISIS420951, ISIS 425653, ISIS 425655, ISIS 425656, ISIS 425679, ISIS 425695,ISIS 425736, ISIS 425737, ISIS 425755, or ISIS 425757. Another group offour female hTTR transgenic mice was injected with 25 mg/kg of controloligonucleotide ISIS 141923 (CCTTCCCTGAAGGTTCCTCC, designated herein asSEQ ID NO: 165) twice a week for four weeks. Another group of four hTTRtransgenic female mice were injected subcutaneously with PBS twice aweek for four weeks. The mice injected with PBS served as a controlgroup. Blood samples were collected from all groups on weeks 0, 1, 2, 3,and 4 for plasma transthyretin level analysis. The mice were sacrificedtwo days after the last dose and livers were harvested for target mRNAanalysis.

RNA analysis

RNA was extracted from liver tissue for real-time PCR analysis oftransthyretin using primer probe set RTS3029. Results are presented aspercent inhibition of human transthyretin, relative to PBS control. Asshown in Table 14, treatment with ISIS antisense oligonucleotidesresulted in significant reduction of human transthyretin mRNA incomparison to the PBS control. Treatment with the controloligonucleotide, ISIS 141923 did not result in significant reduction oftransthyretin, as expected.

TABLE 14 Inhibition of human transthyretin mRNA in the hTTR transgenicmice liver relative to the PBS control ISIS % NO inhibition 304299 79304309 83 304311 63 304312 64 420915 82 420951 92 425653 66 425655 76425656 76 425679 93 425695 82 425736 63 425737 76 425755 91 425757 91141923 28

Protein Analysis

Human transthyretin protein levels were measured in transgenic miceplasma by ELISA using an anti-transthyretin polyclonal antibody (AbcamAb37774) and a sheep anti-TTR horse radish peroxidase detection antibody(Abcam cat. no. 35217). The color reaction was developed by theImmunoPure® TMB Substrate Kit and absorbance measured at 450 nm using amicrotiter plate spectrophotometer. Plasma samples were taken predoseand on days 7, 14 and 28. The results are presented in Table 15expressed as percentage inhibition compared to the predose levels anddemonstrate a time-dependent reduction in protein levels with treatmentwith ISIS oligonucleotides.

TABLE 15 Inhibition of human transthyretin protein in the hTTRtransgenic mice plasma relative to predose levels ISIS ISIS ISIS ISISISIS ISIS ISIS ISIS PBS 304299 304309 420915 420951 425679 425695 425755141923 Day 7 0 50 63 71 92 99 69 57 3 Day 14 3 76 78 90 98 100 80 72 3Day 21 20 88 81 95 100 99 88 78 13 Day 28 13 89 83 98 100 100 91 79 8

Body Weight and Organ Weight

The body weights of the mice were measured predose and at the end of thetreatment period. The body weights are presented in Table 16 and areexpressed as percent increase over the PBS control weight taken beforethe start of treatment. Liver, spleen and kidney weights were measuredat the end of the study, and are also presented in Table 16 as a percentchange over the respective organ weights of the PBS control. As shown inTable 16, there was no significant change in body or organ weights as aresult of antisense oligonucleotide treatment.

TABLE 16 Percent change in body and organ weights of transgenic miceafter antisense oligonucleotide treatment Body weight Liver SpleenKidney PBS 1.1 1.0 1.0 1.0 ISIS 304299 1.1 1.1 1.0 0.8 ISIS 304309 1.11.1 1.0 1.0 ISIS 304311 1.1 1.2 1.0 1.2 ISIS 304312 1.1 1.3 1.0 0.8 ISIS420915 1.1 1.1 1.0 1.1 ISIS 420951 1.1 1.2 1.0 1.5 ISIS 425653 1.1 1.10.9 1.0 ISIS 425655 1.1 1.3 1.0 1.2 ISIS 425656 1.2 1.3 1.0 1.3 ISIS425679 1.2 1.2 1.0 1.6 ISIS 425695 1.1 1.3 1.0 1.0 ISIS 425736 1.2 1.21.0 1.0 ISIS 425737 1.1 1.2 1.1 1.2 ISIS 425755 1.2 1.3 1.1 1.3 ISIS425757 1.1 1.9 1.0 1.5 ISIS 141923 1.1 1.1 1.0 0.8

Liver Function

To evaluate the effect of ISIS oligonucleotides on hepatic function,plasma concentrations of transaminases were measured using an automatedclinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.).Plasma concentrations of ALT (alanine transaminase) and AST (aspartatetransaminase) were measured and the results are presented in Table 17,expressed in IU/L. Plasma levels of bilirubin were also measured usingthe same clinical chemistry analyzer; results are also presented inTable 17 and expressed in mg/dL.

TABLE 17 Effect of antisense oligonucleotide treatment on metabolicmarkers in the liver of transgenic mice ALT AST Bilirubin (IU/L) (IU/L)(mg/dL) PBS 31 78 0.23 ISIS 304299 40 121 0.19 ISIS 304309 38 119 0.20ISIS 304311 34 60 0.16 ISIS 304312 43 67 0.17 ISIS 420915 34 75 0.26ISIS 420951 75 124 0.17 ISIS 425653 35 78 0.20 ISIS 425655 131 109 0.16ISIS 425656 68 110 0.19 ISIS 425679 119 180 0.20 ISIS 425695 43 69 0.15ISIS 425736 23 58 0.16 ISIS 425737 35 64 0.19 ISIS 425755 109 162 0.16ISIS 425757 1904 937 0.24 ISIS 141923 31 76 0.19

Kidney Function

To evaluate the effect of ISIS oligonucleotides on kidney function,plasma concentrations of blood urea nitrogen (BUN) were measured usingan automated clinical chemistry analyzer (Hitachi Olympus AU400e,Melville, N.Y.). Results are presented in Table 18, expressed in mg/dL.The data indicates that antisense inhibition of transthyretin has noeffect on BUN levels in these transgenic mice.

TABLE 18 Effect of antisense oligonucleotide treatment on BUN (mg/dL) inthe kidney of transgenic mice BUN (mg/dL) PBS 26 ISIS 304299 24 ISIS304309 29 ISIS 304311 28 ISIS 304312 26 ISIS 420915 25 ISIS 420951 25ISIS 425653 24 ISIS 425655 28 ISIS 425656 25 ISIS 425679 26 ISIS 42569528 ISIS 425736 25 ISIS 425737 23 ISIS 425755 24 ISIS 425757 25 ISIS141923 23

Example 11 Tolerability of Antisense Oligonucleotides Targeting HumanTransthyretin in CD1 Mice

CD1® mice (Charles River, Mass.) are a multipurpose model of mice,frequently utilized for safety and efficacy testing. The mice weretreated with ISIS antisense oligonucleotides selected from studiesdescribed in Example 10 and evaluated for changes in the levels ofvarious metabolic markers.

Treatment

Groups of eight CD1 mice each were injected subcutaneously twice a weekwith 50 mg/kg of ISIS 304299, ISIS 304309, ISIS 420915, ISIS 420951,ISIS 425655, ISIS 425656, ISIS 425679, ISIS 425695, ISIS 425736, ISIS425737, and ISIS 425755. Four mice from each group were evaluated atweek 2 and week 6 of the treatment period. Three days after the lastdose at each time point, body weights were taken, mice were euthanizedand organs and plasma were harvested for further analysis.

Body and Organ Weights

The body weights of the mice were measured pre-dose and at the end ofeach treatment period (two weeks and six weeks). The body weights arepresented in Tables 19 and 20, and are expressed as percent increaseover the PBS control weight taken before the start of treatment. Liver,spleen and kidney weights were measured at the end of the study, and arealso presented in Tables 19 and 20 as a percentage change over therespective organ weights of the PBS control.

TABLE 19 Change in body and organ weights of CD1 mice after antisenseoligonucleotide treatment (%) at week 2 Body weight Liver Spleen KidneyPBS 1.1 1.0 1.0 1.0 ISIS 304299 1.1 1.1 1.1 1.1 ISIS 304309 1.1 1.1 1.11.0 ISIS 420915 1.1 1.1 1.1 1.0 ISIS 420951 1.1 1.3 1.7 1.2 ISIS 4256551.1 1.2 1.2 0.9 ISIS 425656 1.1 1.1 1.1 1.0 ISIS 425679 1.1 1.1 1.4 1.1ISIS 425695 1.1 1.1 0.9 1.1 ISIS 425736 1.1 1.1 1.0 1.1 ISIS 425737 1.21.1 1.1 1.1 ISIS 425755 1.2 1.2 1.3 1.2

TABLE 20 Change in body and organ weights of CD1 mice after antisenseoligonucleotide treatment (%) at week 6 Body weight Liver Spleen KidneyPBS 1.2 1.0 1.0 1.0 ISIS 304299 1.3 1.2 1.4 1.0 ISIS 304309 1.3 1.3 2.01.0 ISIS 420915 1.3 1.1 1.5 0.9 ISIS 420951 1.3 1.3 2.0 1.1 ISIS 4256551.4 1.3 1.7 0.9 ISIS 425656 1.3 1.3 1.1 1.0 ISIS 425679 1.3 1.4 2.3 1.2ISIS 425695 1.3 1.4 1.5 1.0 ISIS 425736 1.3 1.1 1.2 0.9 ISIS 425737 1.21.1 1.3 1.0 ISIS 425755 1.3 1.3 2.1 1.0

Liver Function

To evaluate the effect of ISIS oligonucleotides on hepatic function,plasma concentrations of transaminases were measured using an automatedclinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.).Plasma concentrations of ALT (alanine transaminase) and AST (aspartatetransaminase) were measured and the results are presented in Tables 21and 22 expressed in IU/L. Plasma levels of bilirubin and albumin werealso measured using the same clinical chemistry analyzer and the resultsare also presented in Tables 21 and 22.

TABLE 21 Effect of antisense oligonucleotide treatment on metabolicmarkers in the liver of CD1 mice at week 2 ALT AST Bilirubin Albumin(IU/L) (IU/L) (mg/dL) (g/dL) PBS 38 66 0.19 5.0 ISIS 304299 42 79 0.333.8 ISIS 304309 52 77 0.22 3.2 ISIS 420915 32 61 0.28 3.5 ISIS 4209511184 804 0.17 3.7 ISIS 425655 60 70 0.20 3.9 ISIS 425656 37 53 0.31 3.5ISIS 425679 88 147 0.23 3.7 ISIS 425695 25 50 0.23 3.6 ISIS 425736 31 790.23 3.2 ISIS 425737 39 43 0.23 3.1 ISIS 425755 104 85 0.29 3.6

TABLE 22 Effect of antisense oligonucleotide treatment on metabolicmarkers in the liver of CD1 mice at week 6 ALT AST Bilirubin Albumin(IU/L) (IU/L) (mg/dL) (g/dL) PBS 31 67 0.20 5.6 ISIS 304299 54 71 0.205.2 ISIS 304309 1211 504 0.30 5.2 ISIS 420915 89 91 0.17 5.0 ISIS 420951872 319 0.20 3.6 ISIS 425655 730 247 0.13 4.3 ISIS 425656 502 261 0.174.3 ISIS 425679 935 475 0.29 4.5 ISIS 425695 1627 563 0.16 4.0 ISIS425736 41 47 0.15 4.1 ISIS 425737 32 55 0.16 4.1 ISIS 425755 233 1760.16 4.3

Kidney Function

To evaluate the effect of ISIS oligonucleotides on kidney function,plasma concentrations of blood urea nitrogen (BUN) and creatinine weremeasured using an automated clinical chemistry analyzer (Hitachi OlympusAU400e, Melville, N.Y.). Results are presented in Tables 23 and 24,expressed in mg/dL.

TABLE 23 Effect of antisense oligonucleotide treatment on metabolicmarkers (mg/dL) in the kidney of CD1 mice at week 2 BUN Creatinine PBS32 0.23 ISIS 304299 26 0.21 ISIS 304309 30 0.19 ISIS 420915 30 0.22 ISIS420951 24 0.17 ISIS 425655 29 0.22 ISIS 425656 25 0.19 ISIS 425679 280.19 ISIS 425695 29 0.19 ISIS 425736 24 0.19 ISIS 425737 24 0.16 ISIS425755 27 0.17

TABLE 24 Effect of antisense oligonucleotide treatment on metabolicmarkers (mg/dL) in the kidney of CD1 mice at week 6 BUN Creatinine PBS24 0.15 ISIS 304299 19 0.11 ISIS 304309 20 0.14 ISIS 420915 24 0.18 ISIS420951 19 0.08 ISIS 425655 22 0.11 ISIS 425656 21 0.10 ISIS 425679 200.06 ISIS 425695 21 0.08 ISIS 425736 22 0.07 ISIS 425737 18 0.07 ISIS425755 22 0.09

Hematology Assays

Blood obtained from all mice groups were sent to Antech Diagnostics forhematocrit (HCT), mean corpuscular volume (MCV), mean corpuscularhemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC)measurements and analyses, as well as measurements of the differentialblood cell counts, such as that of WBC (neutrophils, lymphocytes, andmonocytes), RBC, and platelets, and total hemoglobin content. Theresults are presented in Tables 25-28. Percentages given in the tablesindicate the percent change in total blood cell count compared to thePBS control. Those antisense oligonucleotides which did not affect adecrease in platelet count less than 70% of the PBS control or anincrease in monocyte count more than two-fold were selected for furtherstudies.

TABLE 25 Effect of antisense oligonucleotide treatment on complete bloodcell count (%) compared to the PBS control in CD1 mice at week 2 ISISNO. WBC RBC Hemoglobin HCT MCV MCH MCHC 304299 −15 −3 −2 0 +3 +1 −1304309 −13 −4 −7 −6 −2 −4 −2 420915 +7 −7 −7 −5 +2 +1 −2 420951 +79 −6−5 −5 +1 +1 0 425655 +56 −3 −5 −4 −1 −2 −1 425656 +69 −5 −6 −5 0 −1 −2425679 +30 −6 −7 −7 −1 −1 0 425695 +49 −3 −4 −4 0 0 +1 425736 +15 −6 −6−4 +1 0 −2 425737 +19 −5 −7 −5 −1 −3 −2 425755 +85 −3 −6 −6 −4 −3 0

TABLE 26 Effect of antisense oligonucleotide treatment on complete bloodcell count (%) compared to the PBS control in CD1 mice at week 6 ISISNO. WBC RBC Hemoglobin HCT MCV MCH MCHC 304299 −7 −9 −10 −13 −5 0 +4304309 +10 −12 −11 −15 −5 +1 +6 420915 +11 −7 −8 −10 −4 −2 +2 420951 +81−12 −20 −19 −9 −9 −1 425655 +29 −3 −11 −10 −8 −9 −2 425656 +72 −1 −5 −6−4 −5 −1 425679 +154 −11 −20 −21 −10 −9 +2 425695 +118 +3 −9 −9 −2 −12+3 425736 +51 +4 −5 −7 0 −10 +1 425737 +30 +8 −1 −2 0 −8 +1 425755 +54−1 −11 −12 −8 −10 0

TABLE 27 Effect of antisense oligonucleotide treatment on differentialblood cell count (%) compared to the PBS control in CD1 mice at week 2ISIS NO. Neutrophils Monocytes Lymphocytes Platelets 304299 11 −3 20 17304309 −11 5 8 14 420915 1 4 −24 41 420951 18 −7 32 −9 425655 18 −5 2018 425656 31 −7 −4 24 425679 2 −1 24 −19 425695 −50 15 20 29 425736 8 −10 10 425737 −29 10 −8 24 425755 −13 7 −4 9

TABLE 28 Effect of antisense oligonucleotide treatment on differentialblood cell count (%) compared to the PBS control in CD1 mice at week 6ISIS NO. Neutrophils Lymphocytes Monocytes Platelets 304299 −60 +26 +10−16 304309 −28 +12 +30 +2 420915 −29 +6 +50 −30 420951 −26 +11 0 −40425655 −16 +8 −10 −19 425656 −22 +16 −50 −25 425679 −36 +19 −20 −27425695 −25 +9 −15 −49 425736 −41 +16 −5 −46 425737 −53 +23 −20 −65425755 −20 +4 +25 −41

Example 12 Measurement of Half-Life of Antisense Oligonucleotide in CD1Mouse Liver

CD1 mice were treated with ISIS antisense oligonucleotides from studiesdescribed in Example 11 and the oligonucleotide half-life as well as theelapsed time for oligonucleotide degradation and elimination from theliver was evaluated.

Treatment

Groups of twelve CD1 mice each were injected subcutaneously twice perweek for 2 weeks with 50 mg/kg of ISIS 304299, ISIS 304309, ISIS 420915,ISIS 420951, ISIS 425655, ISIS 425656, ISIS 425679, ISIS 425695, ISIS425736, ISIS 425737, and ISIS 425755. Four mice from each group weresacrificed 3 days, 28 days and 56 days following the final dose. Liverswere harvested for analysis.

Measurement of Oligonucleotide Concentration

The concentration of the full-length oligonucleotide as well as thetotal oligonucleotide concentration (including the degraded form) wasmeasured. The method used is a modification of previously publishedmethods (Leeds et al., 1996; Geary et al., 1999) which consist of aphenol-chloroform (liquid-liquid) extraction followed by a solid phaseextraction. An internal standard (ISIS 355868, a 27-mer2′-O-methoxyethyl modified phosphorothioate oligonucleotide,GCGTTTGCTCTTCTTCTTGCGTTTTTT, designated herein as SEQ ID NO: 166) wasadded prior to extraction. Tissue sample concentrations were calculatedusing calibration curves, with a lower limit of quantitation (LLOQ) ofapproximately 1.14 μg/g. Half-lives were then calculated using WinNonlinsoftware (PHARSIGHT).

The results are presented in Tables 29 and 30, expressed as μg/g livertissue. The half-life of each oligonucleotide is presented in Table 31.Antisense oligonucleotides with half-lives within 11-34 days were chosenfor further studies.

TABLE 29 Full-length oligonucleotide concentration (μg/g) in the liverof CD1 mice ISIS NO. 3 days 28 days 56 days 304299 180 56 8 304309 317254 106 420915 248 126 34 420951 173 109 49 425655 191 113 33 425656 25673 29 425679 201 73 27 425695 315 194 65 425736 219 110 47 425737 190 409 425755 211 120 47

TABLE 30 Total oligonucleotide concentration (μg/g) in the liver of CD1mice ISIS NO. 3 days 28 days 56 days 304299 268 168 38 304309 389 354152 420915 314 229 83 420951 262 196 131 425655 298 217 87 425656 328135 85 425679 333 161 103 425695 364 263 143 425736 298 211 140 425737266 117 31 425755 337 227 140

TABLE 31 Half-life of oligonucleotide (days) in the liver of CD1 miceISIS NO. Half-life (days) 304299 12 304309 33 420915 19 420951 29 42565521 425656 17 425679 18 425695 23 425736 24 425737 12 425755 24

Example 13 Tolerability of Antisense Oligonucleotides Targeting HumanTransthyretin in Sprague-Dawley Rats

Sprague-Dawley rats were treated with ISIS antisense oligonucleotidesselected from studies described in Examples 11 and 12 and evaluated forchanges in the levels of various metabolic markers.

Treatment

The body weights, complete blood count and different blood count, aswell as the urine protein/creatinine ratio of the rats were evaluatedpre-dose. Groups of four Sprague-Dawley rats each were injectedsubcutaneously twice a week with 50 mg/kg of ISIS 304299, ISIS 304309,ISIS 420915, ISIS 420951, ISIS 425655, ISIS 425656, ISIS 425679, ISIS425695, ISIS 425736, ISIS 425737, and ISIS 425755. Three days after thelast dose at each time point, body weights were taken, mice wereeuthanized and organs and plasma were harvested for further analysis.

Body and Organ Weights

The body weights of the rats were measured pre-dose and at the end ofthe treatment period. The body weights are presented in Table 32, andare expressed as percent increase over the PBS control weight takenbefore the start of treatment. Liver, spleen and kidney weights weremeasured at the end of the study, and are also presented in Table 32 asa percentage change over the respective organ weights of the PBScontrol.

TABLE 32 Change in body and organ weights of Sprague-Dawley rats afterantisense oligonucleotide treatment (%) Body weight Liver Spleen KidneyPBS 1.6 1.0 1.0 1.0 ISIS 304299 1.2 1.7 4.9 1.6 ISIS 304309 1.1 1.6 4.31.4 ISIS 420915 1.4 1.4 3.3 1.3 ISIS 420951 1.1 1.4 5.0 1.5 ISIS 4256551.2 1.5 3.4 1.3 ISIS 425656 1.2 1.5 2.9 1.2 ISIS 425679 1.0 1.9 6.4 1.7ISIS 425695 1.2 1.6 3.3 1.3 ISIS 425736 1.3 1.5 2.9 1.2 ISIS 425737 1.21.7 4.0 1.5 ISIS 425755 1.0 1.5 5.4 1.5

As shown in Tables 32, certain compounds showed a less than a 4 -foldincrease in spleen weight.

Liver Function

To evaluate the effect of ISIS oligonucleotides on hepatic function,plasma concentrations of transaminases were measured using an automatedclinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.).Plasma concentrations of ALT (alanine transaminase) and AST (aspartatetransaminase) were measured and the results are presented in Table 33expressed in IU/L. Plasma levels of bilirubin and albumin were alsomeasured using the same clinical chemistry analyzer and the results arealso presented in Table 33.

TABLE 33 Effect of antisense oligonucleotide treatment on metabolicmarkers in the liver of Sprague-Dawley rats ALT AST Bilirubin Albumin(IU/L) (IU/L) (mg/dL) (g/dL) PBS 55 138 0.15 3.3 ISIS 304299 69 154 0.152.7 ISIS 304309 80 138 0.11 2.9 ISIS 420915 43 95 0.11 3.0 ISIS 420951353 511 0.32 2.6 ISIS 425655 312 497 0.47 2.6 ISIS 425656 277 335 0.203.0 ISIS 425679 537 659 0.38 2.7 ISIS 425695 228 445 0.23 2.3 ISIS425736 362 553 0.32 2.9 ISIS 425737 55 79 0.09 1.9 ISIS 425755 271 3030.41 2.8

Kidney Function

To evaluate the effect of ISIS oligonucleotides on kidney function,plasma concentrations of blood urea nitrogen (BUN) and creatinine weremeasured using an automated clinical chemistry analyzer (Hitachi OlympusAU400e, Melville, N.Y.). Results are presented in Table 34, expressed inmg/dL. The ratio of total urine protein to creatinine was also evaluatedand presented in Table 35.

TABLE 34 Effect of antisense oligonucleotide treatment on metabolicmarkers (mg/dL) in the kidney of Sprague-Dawley rats BUN Creatinine PBS20 0.26 ISIS 304299 30 0.40 ISIS 304309 24 0.33 ISIS 420915 20 0.26 ISIS420951 37 0.47 ISIS 425655 28 0.40 ISIS 425656 25 0.34 ISIS 425679 460.42 ISIS 425695 30 0.37 ISIS 425736 26 0.37 ISIS 425737 30 0.36 ISIS425755 29 0.36

TABLE 35 Effect of antisense oligonucleotide treatment on total urineprotein/creatinine in the kidney of Sprague-Dawley rats Pre-dose Week 6PBS 0.82 0.95 ISIS 304299 0.95 7.57 ISIS 304309 1.10 5.20 ISIS 4209150.91 5.30 ISIS 420951 0.90 5.02 ISIS 425655 0.78 6.03 ISIS 425656 0.869.37 ISIS 425679 0.91 7.80 ISIS 425695 0.89 5.71 ISIS 425736 1.00 5.85ISIS 425737 0.86 43.76 ISIS 425755 0.78 3.70

As shown in Tables 34 and 35, certain compounds demonstrated a less than7-fold increase in the total urine protein/creatinine in the kidney ofthese rats. Furthermore, certain compounds demonstrated a less than6-fold increase in the total urine protein/creatinine in the kidney ofthese rats.

Hematology Assays

Blood obtained from all rat groups were sent to Antech Diagnostics forhematocrit (HCT), mean corpuscular volume (MCV), mean corpuscularhemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC)measurements and analyses, as well as measurements of the differentialblood cell counts, such as that of WBC (neutrophils, lymphocytes, andmonocytes), RBC, and platelets, and total hemoglobin content. Theresults are presented in Tables 36 and 37. Percentages given in thetables indicate the percent change in total blood cell count compared tothe PBS control.

TABLE 36 Effect of antisense oligonucleotide treatment on complete bloodcell count (%) compared to the PBS control in Sprague-Dawley rats ISISNO. WBC RBC Hemoglobin HCT MCV MCH MCHC 304299 +4 −5 −3 +2 +11 +5 −5304309 −10 −8 −11 −12 −4 −3 +1 420915 −9 −16 −20 −17 +1 −3 −3 420951 +5−5 −8 −5 +1 −2 −3 425655 +22 −17 −18 −19 −2 0 +2 425656 −1 −13 −19 −16−3 −6 −2 425679 +49 −42 −32 −28 +26 +19 −5 425695 −2 −25 −31 −29 −4 −8−3 425736 +18 +1 −3 +2 0 −4 −4 425737 −15 −20 −18 −20 +2 +3 +1 425755+35 −31 −27 −23 +14 +8 −4

TABLE 37 Effect of antisense oligonucleotide treatment on complete bloodcell count (%) compared to the PBS control in Sprague-Dawley rats ISISNO. Neutrophils Lymphocytes Monocytes Platelet 304299 −61 +15 −10 −41304309 −35 +8 +10 −37 420915 −23 +6 0 −29 420951 −62 +15 +10 −67 425655+23 −8 +80 −13 425656 −14 0 +70 −15 425679 −4 −1 +60 −75 425695 +68 −20+80 −5 425736 0 −2 +70 −1 425737 −6 +1 +20 −21 425755 −18 +3 +70 −58

Example 14 Pharmacokinetic Studies of Antisense OligonucleotideConcentration in Sprague-Dawley Rat Liver and Kidney

Sprague Dawley rats were treated with ISIS antisense oligonucleotidesfrom studies described in Example 13 and the oligonucleotide half-lifeas well as the elapsed time for oligonucleotide degradation andelimination from the liver and kidney was evaluated.

Treatment

Groups of four Sprague Dawley rats each were injected subcutaneouslytwice a week for 2 weeks with 20 mg/kg of ISIS 304299, ISIS 304309, ISIS420915, ISIS 420951, ISIS 425655, ISIS 425656, ISIS 425679, ISIS 425695,ISIS 425736, ISIS 425737, and ISIS 425755. Three days after the lastdose, the rats were sacrificed and livers and kidneys were collected foranalysis.

Measurement of Oligonucleotide Concentration

The concentration of the full-length oligonucleotide as well as thetotal oligonucleotide concentration (including the degraded form) wasmeasured. The method used is a modification of previously publishedmethods (Leeds et al., 1996; Geary et al., 1999) which consist of aphenol-chloroform (liquid-liquid) extraction followed by a solid phaseextraction. An internal standard (ISIS 355868, a 27-mer2′—O-methoxyethyl modified phosphorothioate oligonucleotide,GCGTTTGCTCTTCTTCTTGCGTTTTTT, designated herein as SEQ ID NO: 166) wasadded prior to extraction. Tissue sample concentrations were calculatedusing calibration curves, with a lower limit of quantitation (LLOQ) ofapproximately 1.14 μg/g. The results are presented in Tables 38 and 39,expressed as μg/g liver or kidney tissue. The kidney to liver ratio offull length oligonucleotide was also calculated and presented in Table38.

TABLE 38 Full-length oligonucleotide concentration (μg/g) and ratio inthe liver and kidney of Sprague-Dawley rats Kidney/Liver ISIS NO. LiverKidney Ratio 304299 165 487 2.9 304309 344 606 1.8 420915 171 680 4.0420951 214 389 1.8 425655 242 466 1.9 425656 286 595 2.1 425679 290 3341.2 425695 266 566 2.1 425736 245 571 2.3 425737 167 477 2.9 425755 218379 1.7

TABLE 39 Total oligonucleotide concentration (μg/g) in the liver andkidney of Sprague-Dawley rats ISIS NO. Liver Kidney 304299 208 653304309 409 803 420915 196 844 420951 348 879 425655 340 764 425656 329703 425679 461 710 425695 369 843 425736 282 738 425737 195 587 425755351 886

Example 15 In Vivo Dose-Dependent Inhibition of Human Transthyretin inTransgenic Mice

Transgenic mice containing the human transthyretin gene were dosed inincreasing doses of ISIS oligonucleotides selected from studiesdescribed in Example 14 to evaluate the effect of dose-dependentinhibition of human transthyretin in these mice.

Treatment

Groups of four mice, two male and two female, each were injectedsubcutaneously twice a week for 4 weeks with 4 mg/kg, 10 mg/kg or 25mg/kg of ISIS 304299, ISIS 420915, ISIS 420951, ISIS 425679, ISIS425736, ISIS 425737, or ISIS 425755. One group of four mice, two maleand two female, was injected subcutaneously twice a week for 4 weekswith 25 mg/kg of the control oligonucleotide, ISIS 141923. One controlgroup of four mice, two male and two female, was injected subcutaneouslytwice a week for 4 weeks with PBS. Plasma samples were taken from eachgroup at days 0, 7, 14, 21 and 28. Two days after the last dose, themice were euthanized and organs were harvested for further analysis.

RNA Analysis

RNA was extracted from liver tissue for real-time PCR analysis oftransthyretin using primer probe set RTS3029. Results are presented aspercent inhibition of human transthyretin, relative to PBS control. Asshown in Table 40, treatment with ISIS antisense oligonucleotidesresulted in significant dose-dependent reduction of human transthyretinmRNA in comparison to the PBS control. Treatment with the controloligonucleotide, ISIS 141923 did not result in significant reduction oftransthyretin, as expected.

TABLE 40 Inhibition of human transthyretin mRNA in the hTTR transgenicmice liver relative to the PBS control ISIS NO. Dose (mg/kg) %inhibition 304299 25 73 10 60 4 9 420915 25 78 10 57 4 43 420951 25 9110 85 4 52 425679 25 94 10 88 4 42 425736 25 49 10 54 4 15 425737 25 8210 59 4 21 425755 25 91 10 79 4 24 141923 25 0

Protein Analysis

Human transthyretin protein levels were measured in transgenic miceplasma by ELISA using an anti-transthyretin polyclonal antibody (AbcamAb37774) and a sheep anti-TTR horse radish peroxidase detection antibody(Abcam cat. no. 35217). The color reaction was developed by theImmunoPure® TMB Substrate Kit and absorbance measured at 450 nm using amicrotiter plate spectrophotometer. Plasma samples were taken predoseand on days 7, 14, 21 and 28. The results are presented in Table 41expressed as percentage inhibition compared to the predose levels anddemonstrate a time-dependent and dose-dependent reduction in proteinlevels on treatment with ISIS oligonucleotides.

TABLE 41 Inhibition of human transthyretin protein in transgenic miceplasma relative to pre-dose levels ISIS Day Day Day Day Day NO. 0 7 1421 28 141923 25 0 0 20 77 41 304299 25 0 44 85 100 88 10 0 0 8 93 78 4 00 0 57 0 420915 25 0 0 67 86 91 10 0 21 39 70 71 4 0 25 0 0 0 420951 250 83 96 100 100 10 0 35 66 91 86 4 0 7 26 0 0 425679 25 0 93 97 96 98 100 38 80 96 95 4 0 0 0 0 0 425736 25 0 56 76 82 92 10 0 0 33 37 66 4 0 00 0 0 425737 25 0 90 96 99 98 10 0 51 80 88 89 4 0 29 21 37 31 425755 250 88 96 98 99 10 0 52 76 90 88 4 0 29 22 36 26

Body Weight and Organ Weight

The body weights of the mice were measured pre-dose and at the end ofthe treatment period. The body weights are presented in Table 42 and areexpressed as percent increase over the PBS control weight taken beforethe start of treatment. Liver, spleen and kidney weights were measuredat the end of the study, and are also presented in Table 42 as apercentage change over the respective organ weights of the PBS control.

TABLE 42 Change in body and organ weights of transgenic mice afterantisense oligonucleotide treatment (%) Dose (mg/kg) Body weight LiverSpleen Kidney PBS +13 0 0 0 ISIS 25 +17 +16 +3 −2 304299 10 +14 +10 −13−4 4 +17 +2 +17 −2 ISIS 25 +18 +12 −6 −6 420915 10 +16 +6 −4 −5 4 +15 +4+8 −2 ISIS 25 +22 +23 +32 −2 420951 10 +16 +11 +10 −3 4 +24 +7 +19 +5ISIS 25 +24 +33 +40 −1 425679 10 +14 +5 +9 −2 4 +19 +7 +10 0 ISIS 25 +16+15 0 −5 425736 10 +28 +8 −12 −6 4 +20 +10 −9 −2 ISIS 25 +16 +13 0 −2425737 10 +19 +6 +18 −3 4 +19 +5 +4 +1 ISIS 25 +21 +25 +34 −5 425755 10+17 +10 +13 −4 4 +22 +3 +27 +4 ISIS 25 +20 +8 −3 −4 141923Liver function

To evaluate the effect of ISIS oligonucleotides on hepatic function,plasma concentrations of transaminases were measured using an automatedclinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.).Plasma concentrations of ALT (alanine transaminase) and AST (aspartatetransaminase) were measured and the results are presented in Table 43expressed in IU/L. Plasma levels of bilirubin were also measured usingthe same clinical chemistry analyzer; results are also presented inTable 43 and expressed in mg/dL.

TABLE 43 Effect of antisense oligonucleotide treatment on metabolicmarkers in the liver of transgenic mice Dose ALT AST TBIL (mg/kg) (IU/L)(IU/L) (mg/dL) PBS — 48 112 0.20 ISIS 25 42 93 0.14 304299 10 37 56 0.184 35 71 0.15 ISIS 25 63 181 0.22 420915 10 46 132 0.22 4 35 114 0.22ISIS 25 63 85 0.17 420951 10 42 107 0.21 4 31 74 0.19 ISIS 25 156 1500.13 425679 10 93 148 0.23 4 38 119 0.22 ISIS 25 37 78 0.21 425736 10 3362 0.20 4 46 228 0.23 ISIS 25 55 121 0.20 425737 10 41 94 0.18 4 32 730.14 ISIS 25 74 160 0.17 425755 10 31 80 0.16 4 45 122 0.21 ISIS 25 66141 0.17 141923

Kidney Function

To evaluate the effect of ISIS oligonucleotides on kidney function,plasma concentrations of blood urea nitrogen (BUN) were measured usingan automated clinical chemistry analyzer (Hitachi Olympus AU400e,Melville, N.Y.). Results are presented in Table 44, expressed in mg/dL.

TABLE 44 Effect of antisense oligonucleotide treatment on BUN (mg/dL) inthe kidney of transgenic mice Dose (mg/kg) BUN PBS 22 ISIS 25 22 30429910 22 4 22 ISIS 25 24 420915 10 25 4 20 ISIS 25 24 420951 10 25 4 26ISIS 25 26 425679 10 24 4 22 ISIS 25 20 425736 10 22 4 22 ISIS 25 21425737 10 19 4 23 ISIS 25 23 425755 10 21 4 20 ISIS 25 21 141923

Example 16 In Vivo Inhibition of Human Transthyretin in HumanTransthyretin-Transgenic Mice

Antisense oligonucleotides with 5-10-5 MOE motifs, ISIS 304313, ISIS420913, ISIS 420919, ISIS 420921, ISIS 420922, ISIS 420937, ISIS 420944,ISIS 420947, ISIS 420949, ISIS 420950, ISIS 420951, ISIS 420952, ISIS420953, ISIS 420955, ISIS 420957, and ISIS 420959 from Table 4. Theseantisense oligonucleotides exhibited 65% inhibition or more oftransthyretin mRNA were selected and tested in transgenic micecontaining the human transthyretin gene. Additional oligonucleotideswith overlapping sequences to ISIS 420951 (GTTTTATTGTCTCTGCCTGG (SEQ IDNO: 116)), and with various motifs were also designed to test in thetransgenic mice. These additional oligonucleotides were ISIS 450518(TTTTATTGTCTCTGCCTG (SEQ ID NO: 5-8-5 MOE (SEQ ID NO: 167)), ISIS 450519(GTTTTATTGTCTCTGCCTGG, 6-8-6 MOE (SEQ ID NO: 116)), ISIS 450520(GTTTTATTGTCTCTGCCTGG, 3-10-7 MOE (SEQ ID NO: 116)), ISIS 450521(GTTTTATTGTCTCTGCCTGG, 7-10-3 MOE (SEQ ID NO: 116)), ISIS 450522(GTTTTATTGTCTCTGCCTGG, 2-10-8 MOE (SEQ ID NO: 116)), and ISIS 450523(GTTTTATTGTCTCTGCCTGG, 8-10-2 MOE (SEQ ID NO: 116)).

Treatment

Groups of four hTTR transgenic mice each, two male and two female, wereadministered subcutaneously twice per week for four weeks with 25 mg/kgof ISIS 304313, ISIS 420913, ISIS 420919, ISIS 420921, ISIS 420922, ISIS420937, ISIS 420944, ISIS 420947, ISIS 420949, ISIS 420950, ISIS 420951,ISIS 420952, ISIS 420953, ISIS 420955, ISIS 420957, ISIS 420959, ISIS425518, ISIS 425519, ISIS 425520, ISIS 425521, ISIS 425522, or ISIS425523. A control group four hTTR transgenic mice, two male and twofemale, were injected subcutaneously with PBS twice per week for fourweeks. Blood samples were collected from all groups on days 0, 14 and 28for plasma transthyretin level analysis. The mice were sacrificed twodays after the last dose and livers were harvested for target mRNAanalysis.

RNA Analysis

RNA was extracted from liver tissue for real-time PCR analysis oftransthyretin using primer probe set RTS3029. Results are presented aspercent inhibition of human transthyretin, relative to PBS control. Asshown in Table 45, treatment with ISIS antisense oligonucleotidesresulted in significant reduction of human transthyretin mRNA incomparison to the PBS control.

TABLE 45 Inhibition of human transthyretin mRNA in the hTTR transgenicmice liver relative to the PBS control ISIS NO. % inhibition 304313 68420913 83 420919 64 420921 70 420922 82 420937 46 420944 58 420947 62420949 87 420950 94 420952 95 420953 93 420955 93 420957 90 420959 73450518 80 450519 87 450520 85 450521 94 450522 73 450523 94 420951 94

Protein Analysis

Human transthyretin protein levels were measured in transgenic miceplasma by ELISA using an anti-transthyretin transthyretin polyclonalantibody (Abcam Ab37774) and a sheep anti-TTR horse radish peroxidasedetection antibody (Abcam cat. no. 35217). The color reaction wasdeveloped by the ImmunoPure® TMB Substrate Kit and absorbance measuredat 450 nm using a microtiter plate spectrophotometer. Plasma sampleswere taken predose and on days 7, 14 and 28. The results are presentedin Table 46 expressed as percentage inhibition compared to the pre-doselevels and demonstrate a time-dependent reduction in protein levels ontreatment with ISIS oligonucleotides.

TABLE 46 Inhibition of human transthyretin protein in the hTTRtransgenic mice plasma relative to pre-dose levels Day 0 Day 14 Day 28PBS 0 0 0 ISIS 304313 0 62 77 ISIS 420913 0 91 97 ISIS 420919 0 70 82ISIS 420921 0 83 87 ISIS 420922 0 95 97 ISIS 420937 0 37 59 ISIS 4209440 57 72 ISIS 420947 0 57 65 ISIS 420949 0 93 99 ISIS 420950 0 97 100ISIS 420952 0 98 100 ISIS 420953 0 99 100 ISIS 420955 0 89 100 ISIS420957 0 92 94 ISIS 420959 0 69 87 ISIS 450518 0 80 97 ISIS 450519 0 94100 ISIS 450520 0 83 100 ISIS 450521 0 100 100 ISIS 450522 0 93 97 ISIS450523 0 100 100 ISIS 420951 0 99 100

Liver Function

To evaluate the effect of ISIS oligonucleotides on hepatic function,plasma concentrations of transaminases were measured using an automatedclinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.).Plasma concentrations of ALT (alanine transaminase) and AST (aspartatetransaminase) were measured and the results are presented in Table 47expressed in IU/L. Plasma levels of bilirubin were also measured usingthe same clinical chemistry analyzer; results are also presented inTable 47 and expressed in mg/dL.

TABLE 47 Effect of antisense oligonucleotide treatment on metabolicmarkers in the liver of transgenic mice ALT AST Bilirubin (IU/L) (IU/L)(mg/dL) PBS 34 88 0.20 ISIS 304313 42 79 0.16 ISIS 420913 35 67 0.17ISIS 420919 63 177 0.20 ISIS 420921 47 103 0.15 ISIS 420922 42 128 0.16ISIS 420937 33 160 0.15 ISIS 420944 38 84 0.15 ISIS 420947 42 120 0.17ISIS 420949 46 125 0.15 ISIS 420950 73 106 0.15 ISIS 420952 151 271 0.19ISIS 420953 982 452 0.16 ISIS 420955 47 80 0.15 ISIS 420957 53 133 0.18ISIS 420959 31 89 0.11 ISIS 450518 103 200 0.20 ISIS 450519 64 81 0.12ISIS 450520 350 270 0.12 ISIS 450521 104 226 0.13 ISIS 450522 109 2010.14 ISIS 450523 80 170 0.19 ISIS 420951 67 100 0.09

Kidney Function

To evaluate the effect of ISIS oligonucleotides on kidney function,plasma concentrations of blood urea nitrogen (BUN) were measured usingan automated clinical chemistry analyzer (Hitachi Olympus AU400e,Melville, N.Y.). Results are presented in Table 48, expressed in mg/dL.

TABLE 48 Effect of antisense oligonucleotide treatment on BUN (mg/dL) inthe kidney of transgenic mice PBS 35 ISIS 304313 29 ISIS 420913 30 ISIS420919 29 ISIS 420921 29 ISIS 420922 27 ISIS 420937 29 ISIS 420944 27ISIS 420947 26 ISIS 420949 25 ISIS 420950 34 ISIS 420952 23 ISIS 42095334 ISIS 420955 24 ISIS 420957 23 ISIS 420959 29 ISIS 450518 28 ISIS450519 25 ISIS 450520 29 ISIS 450521 24 ISIS 450522 29 ISIS 450523 27ISIS 420951 25

Example 17 Tolerability of Antisense Oligonucleotides Targeting HumanTransthyretin in CD1 Mice

CD1 mice were treated with ISIS antisense oligonucleotides from Example16 and evaluated for changes in the levels of various metabolic markers.

Treatment

Groups of eight CD1 mice each were injected subcutaneously twice a weekwith 50 mg/kg of ISIS 304313, ISIS 420913, ISIS 420919, ISIS 420921,ISIS 420922, ISIS 420937, ISIS 420944, ISIS 420947, ISIS 420949, ISIS420950, ISIS 420951, ISIS 420952, ISIS 420953, ISIS 420955, ISIS 420957,ISIS 420959, ISIS 425518, ISIS 425519, ISIS 425520, ISIS 425521, ISIS425522, or ISIS 425523. Three days after the last dose at each timepoint, body weights were taken, mice were euthanized and organs andplasma were harvested for further analysis.

Body and Organ Weights

The body weights of the mice were measured pre-dose and at the end ofeach treatment period (two weeks and six weeks). The body weights arepresented in Table 49 and are expressed as percent increase over the PBScontrol weight taken before the start of treatment. Liver, spleen andkidney weights were measured at the end of the study, and are alsopresented in Table 49 as a percentage change over the respective organweights of the PBS control.

TABLE 49 Change in body and organ weights of CD1 mice after antisenseoligonucleotide treatment (%) at week 6 Body weight Liver Spleen KidneyPBS 1.3 1.0 1.0 1.0 ISIS 304313 1.2 1.2 1.4 1.2 ISIS 420913 1.2 1.2 1.31.1 ISIS 420919 1.3 1.2 1.9 1.1 ISIS 420921 1.1 1.1 2.2 1.1 ISIS 4209221.1 1.0 1.6 0.9 ISIS 420937 1.1 1.0 1.2 1.0 ISIS 420944 1.1 1.1 2.0 1.0ISIS 420947 1.3 1.2 1.7 1.0 ISIS 420949 1.3 1.2 1.8 1.1 ISIS 420950 1.31.0 1.7 1.0 ISIS 420952 1.4 1.3 2.1 0.9 ISIS 420953 1.3 1.5 2.2 1.0 ISIS420955 1.2 1.2 2.2 1.0 ISIS 420957 1.1 1.1 1.8 1.1 ISIS 420959 1.3 1.23.2 1.1 ISIS 450518 1.4 1.3 1.8 1.1 ISIS 450519 1.3 1.5 2.4 1.0 ISIS450520 1.4 1.4 2.2 1.0 ISIS 450521 1.2 1.2 1.9 1.1 ISIS 450522 1.3 1.52.3 1.1 ISIS 450523 1.2 1.3 2.4 1.1 ISIS 420951 1.3 1.2 1.9 1.0

Liver Function

To evaluate the effect of ISIS oligonucleotides on hepatic function,plasma concentrations of transaminases were measured using an automatedclinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.).Plasma concentrations of ALT (alanine transaminase) and AST (aspartatetransaminase) were measured and the results are presented in Table 50expressed in IU/L. Plasma levels of bilirubin and albumin were alsomeasured using the same clinical chemistry analyzer and the results arealso presented in Table 50.

TABLE 50 Effect of antisense oligonucleotide treatment on metabolicmarkers in the liver of CD1 mice ALT AST TBIL PBS 34 88 0.20 ISIS 30431342 79 0.16 ISIS 420913 35 67 0.17 ISIS 420919 63 177 0.20 ISIS 420921 47103 0.15 ISIS 420922 42 128 0.16 ISIS 420937 33 160 0.15 ISIS 420944 3884 0.15 ISIS 420947 42 120 0.17 ISIS 420949 46 125 0.15 ISIS 420950 73106 0.15 ISIS 420952 151 271 0.19 ISIS 420953 982 452 0.16 ISIS 42095547 80 0.15 ISIS 420957 53 133 0.18 ISIS 420959 31 89 0.11 ISIS 450518103 200 0.20 ISIS 450519 64 81 0.12 ISIS 450520 350 270 0.12 ISIS 450521104 226 0.13 ISIS 450522 109 201 0.14 ISIS 450523 80 170 0.19 ISIS420951 67 100 0.09

Kidney Function

To evaluate the effect of ISIS oligonucleotides on kidney function,plasma concentrations of blood urea nitrogen (BUN) and creatinine weremeasured using an automated clinical chemistry analyzer (Hitachi OlympusAU400e, Melville, N.Y.). Results are presented in Table 51, expressed inmg/dL.

TABLE 51 Effect of antisense oligonucleotide treatment on BUN (mg/dL) inthe kidney of CD1 mice BUN PBS 35 ISIS 304313 29 ISIS 420913 30 ISIS420919 29 ISIS 420921 29 ISIS 420922 27 ISIS 420937 29 ISIS 420944 27ISIS 420947 26 ISIS 420949 25 ISIS 420950 34 ISIS 420952 23 ISIS 42095334 ISIS 420955 24 ISIS 420957 23 ISIS 420959 29 ISIS 450518 28 ISIS450519 25 ISIS 450520 29 ISIS 450521 24 ISIS 450522 29 ISIS 450523 27ISIS 420951 25

Hematology Assays

Blood obtained from all mice groups were sent to Antech Diagnostics forhematocrit (HCT), mean corpuscular volume (MCV), mean corpuscularhemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC)measurements and analyses, as well as measurements of the differentialblood cell counts, such as that of WBC (neutrophils, lymphocytes, andmonocytes), RBC, and platelets, and total hemoglobin content. Theresults are presented in Table 52 and 53. Percentages given in thetables indicate the percent change in total blood cell count compared tothe PBS control.

TABLE 52 Effect of antisense oligonucleotide treatment on complete bloodcell count (%) compared to the PBS control in CD1 mice WBC RBCHemoglobin HCT MCV MCH MCHC ISIS 304313 +80 −5 −7 −9 −4 −2 +4 ISIS420913 −10 −1 −3 −5 −4 −2 +3 ISIS 420919 +26 −2 −7 −9 −7 −5 +4 ISIS420921 +60 −9 −12 −15 −6 −3 +5 ISIS 420922 +18 −6 −11 −16 −11 −6 +6 ISIS420937 +42 −3 −4 −7 −5 −1 +5 ISIS 420944 +49 −5 −9 −13 −8 −4 +6 ISIS420947 +36 −2 −2 −5 −3 0 +4 ISIS 420949 +61 −4 −6 −9 −7 −3 +5 ISIS420950 +56 −14 −16 −19 −7 −3 +6 ISIS 420952 +36 −20 −24 −25 −7 −5 +4ISIS 420953 +105 −21 −24 −26 −6 −4 +4 ISIS 420955 +107 −14 −19 −21 −9 −5+6 ISIS 420957 +79 −5 −10 −13 −9 −6 +5 ISIS 420959 +92 −8 −14 −18 −11 −7+6 ISIS 450518 +138 −5 −10 −12 −7 −4 +4 ISIS 450519 +118 −17 −21 −24 −9−5 +6 ISIS 450520 +151 −18 −21 −23 −7 −4 +4 ISIS 450521 +118 −15 −21 −23−11 −7 +5 ISIS 450522 +63 −22 −28 −31 −12 −8 +6 ISIS 450523 +116 −22 −27−29 −11 −7 +6 ISIS 420951 +54 −15 −21 −24 −10 −6 +5

TABLE 53 Effect of antisense oligonucleotide treatment on differentialblood cell count (%) compared to the PBS control in CD1 mice NeutrophilsLymphocytes Monocytes Platelets ISIS 304313 −54 +49 −45 +36 ISIS 420913−46 +39 −21 −2 ISIS 420919 −57 +49 −21 +19 ISIS 420921 −55 +47 −24 +25ISIS 420922 −53 +46 −31 +24 ISIS 420937 −63 +57 −48 +20 ISIS 420944 −40+37 −28 +18 ISIS 420947 −55 +49 −38 −9 ISIS 420949 −30 +24 +7 +17 ISIS420950 −50 +40 0 +6 ISIS 420952 −34 +33 −28 +13 ISIS 420953 −37 +35 −34+11 ISIS 420955 −37 +34 −21 +30 ISIS 420957 −71 +61 −28 +16 ISIS 420959−52 +45 −24 −1 ISIS 450518 −56 +49 −28 +18 ISIS 450519 −18 +11 +41 +55ISIS 450520 −41 +34 0 +7 ISIS 450521 −41 +36 −14 +21 ISIS 450522 −41 +31+17 +58 ISIS 450523 −28 +19 +31 +51 ISIS 420951 −28 +24 0 +26

Example 18 Tolerability of Antisense Oligonucleotides Targeting HumanTransthyretin in Sprague-Dawley Rats

ISIS oligonucleotides selected from studies described in Example 17 werealso tested in Sprague-Dawley rats and evaluated for changes in thelevels of various metabolic markers.

Treatment

The body weights, complete blood count and different blood count, aswell as the urine protein/creatinine ratio of the rats were evaluatedpre-dose. Groups of four Sprague-Dawley rats each were injectedsubcutaneously twice a week with 50 mg/kg of ISIS 420913, ISIS 420921,ISIS 420922, ISIS 420950, ISIS 420955, ISIS 420957, and ISIS 420959.Three days after the last dose at each time point, body weights weretaken, mice were euthanized and organs and plasma were harvested forfurther analysis.

Body and Organ Weights

The body weights of the rats were measured pre-dose and at the end ofthe treatment period. The body weights are presented in Table 54, andare expressed as percent increase over the PBS control weight takenbefore the start of treatment. Liver, spleen and kidney weights weremeasured at the end of the study, and are also presented in Table 54 asa percentage change over the respective organ weights of the PBScontrol.

TABLE 54 Change in body and organ weights of Sprague-Dawley rats afterantisense oligonucleotide treatment (%) Body weight Liver Spleen KidneyPBS 2.1 1.0 1.0 1.0 ISIS 420913 1.5 1.5 4.7 1.1 ISIS 420921 1.6 1.5 4.21.3 ISIS 420922 1.3 1.5 4.4 1.4 ISIS 420950 1.4 1.5 6.4 1.7 ISIS 4209551.5 1.6 5.9 1.4 ISIS 420957 1.4 1.4 6.8 1.3 ISIS 420959 1.5 1.4 5.5 1.4

As shown in Table 54, the compounds demonstrated a less than 10-foldincrease in organ weight of these rats. Furthermore, certain compoundsdemonstrated a less than 7-fold increase in organ weight of these rats.While certain compounds demonstrated a less than 6-fold increase inorgan weight of these rats. Certain compounds demonstrated a less than5-fold increase in organ weight of these rats.

Liver Function

To evaluate the effect of ISIS oligonucleotides on hepatic function,plasma concentrations of transaminases were measured using an automatedclinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.).Plasma concentrations of ALT (alanine transaminase) and AST (aspartatetransaminase) were measured and the results are presented in Table 55expressed in IU/L. Plasma levels of bilirubin and albumin were alsomeasured using the same clinical chemistry analyzer and the results arealso presented in Table 55.

TABLE 55 Effect of antisense oligonucleotide treatment on metabolicmarkers in the liver of Sprague-Dawley rats ALT AST TBIL Albumin (IU/L(IU/L) (mg/dL) (g/dL) PBS 26 66 0.09 4.5 ISIS 420913 38 95 0.08 3.3 ISIS420921 65 151 0.11 3.2 ISIS 420922 40 121 0.11 4.0 ISIS 420950 398 3270.19 4.0 ISIS 420955 78 241 0.18 4.1 ISIS 420957 84 244 0.14 3.7 ISIS420959 82 405 0.17 4.6

Kidney Function

To evaluate the effect of ISIS oligonucleotides on kidney function,plasma concentrations of blood urea nitrogen (BUN) and creatinine weremeasured using an automated clinical chemistry analyzer (Hitachi OlympusAU400e, Melville, NY). Results are presented in Table 56, expressed inmg/dL. The ratio of total urine protein to creatinine was also evaluatedand presented in Table 56.

TABLE 56 Effect of antisense oligonucleotide treatment on metabolicmarkers (mg/dL) in the kidney of Sprague-Dawley rats BUN Creatinine PBS14 0.05 ISIS 420913 22 0.09 ISIS 420921 23 0.07 ISIS 420922 21 0.08 ISIS420950 20 0.11 ISIS 420955 22 0.06 ISIS 420957 23 0.18 ISIS 420959 240.17

TABLE 57 Effect of antisense oligonucleotide treatment on total urineprotein/creatinine in the kidney of Sprague-Dawley rats Urineprotein/creatinine ratio PBS 1.50 ISIS 420913 19.51 ISIS 420921 5.07ISIS 420922 4.72 ISIS 420950 5.61 ISIS 420955 5.57 ISIS 420957 5.40 ISIS420959 4.39

Hematology Assays

Blood obtained from all rat groups were sent to Antech Diagnostics forhematocrit (HCT), mean corpuscular volume (MCV), mean corpuscularhemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC)measurements and analyses, as well as measurements of the differentialblood cell counts, such as that of WBC (neutrophils, lymphocytes, andmonocytes), RBC, and platelets, and total hemoglobin content. Theresults are presented in Tables 58 and 59. Percents given in the tablesindicate the percent change in total blood cell count compared to thePBS control.

TABLE 58 Effect of antisense oligonucleotide treatment on complete bloodcell count (%) compared to the PBS control in Sprague-Dawley rats WBCRBC Hemoglobin HCT MCV MCH MCHC PBS 1.0 1.0 1.0 1.0 1.0 1.0 1.0 ISIS420913 1.7 0.9 0.9 0.9 0.9 0.9 1.0 ISIS 420921 1.6 0.9 0.9 0.9 1.0 1.01.0 ISIS 420922 1.6 0.9 0.9 0.8 1.0 1.0 1.0 ISIS 420950 2.2 0.7 0.7 0.71.0 1.0 1.0 ISIS 420955 1.9 0.7 0.8 0.7 1.1 1.2 1.0 ISIS 420957 3.1 0.80.8 0.8 1.0 1.0 1.0 ISIS 420959 2.2 0.8 0.8 0.8 1.0 1.0 1.0

TABLE 59 Effect of antisense oligonucleotide treatment on differentialblood cell count (%) compared to the PBS control in Sprague-Dawley ratsNeutrophils Lymphocytes Monocytes Platelet PBS 1.0 1.0 1.0 1.0 ISIS420913 0.5 1.1 1.7 0.7 ISIS 420921 0.7 1.0 1.6 0.6 ISIS 420922 0.5 1.11.3 0.7 ISIS 420950 0.8 1.0 2.3 0.7 ISIS 420955 0.5 1.0 2.4 0.7 ISIS420957 0.7 1.0 1.6 0.3 ISIS 420959 0.5 1.1 1.3 n.d.

Example 19 Pharmacokinetic Studies of Half-Life of AntisenseOligonucleotide Concentration in Sprague-Dawley Rat Liver and Kidney

Sprague Dawley rats were treated with ISIS antisense oligonucleotidestargeting from studies described in Example 18 and the oligonucleotidehalf-life as well as the elapsed time for oligonucleotide degradationand elimination from the liver and kidney was evaluated.

Treatment

Groups of four Sprague Dawley rats each were injected subcutaneouslytwice a week for 2 weeks with 20 mg/kg of ISIS 420913, ISIS 420921, ISIS420922, ISIS 420950, ISIS 420955, ISIS 420957, and ISIS 420959. Threedays after the last dose, the rats were sacrificed and livers andkidneys were collected for analysis.

Measurement of Oligonucleotide Concentration

The concentration of the full-length oligonucleotide as well as thetotal oligonucleotide concentration (including the degraded form) wasmeasured. The method used is a modification of previously publishedmethods (Leeds et al., 1996; Geary et al., 1999) which consist of aphenol-chloroform (liquid-liquid) extraction followed by a solid phaseextraction. An internal standard (ISIS 355868, a 27-mer2′-O-methoxyethyl modified phosphorothioate oligonucleotide,GCGTTTGCTCTTCTTCTTGCGTTTTTT, designated herein as SEQ ID NO: 166) wasadded prior to extraction. Tissue sample concentrations were calculatedusing calibration curves, with a lower limit of quantitation (LLOQ) ofapproximately 1.14 μg/g. The results are presented in Tables 60 and 61,expressed as μg/g liver or kidney tissue. The kidney to liver ratio ofoligonucleotide concentration was also calculated and presented inTables 60 and 61.

TABLE 60 Full-length oligonucleotide concentration (μg/g) and ratio inthe liver and kidney of Sprague-Dawley rats Kidney/Liver ISIS NO. LiverKidney ratio 420913 154 285 1.9 420921 147 293 2.0 420922 226 497 2.2420950 161 411 2.6 420955 152 383 2.5 420957 235 453 1.9 420959 187 5132.7

TABLE 61 Total oligonucleotide concentration (μg/g) in the liver andkidney of Sprague-Dawley rats Kidney/Liver ISIS NO. Liver Kidney ratio420913 180 310 1.7 420921 159 305 1.9 420922 238 544 2.3 420950 168 4662.8 420955 156 442 2.8 420957 244 551 2.3 420959 202 534 2.6

Example 20 In Vivo Dose-Dependent Inhibition of Human Transthyretin inTransgenic Mice

ISIS 420913, ISIS 420921, ISIS 420922, ISIS 420957 and ISIS 420959,which exhibited good efficacy and tolerability, as demonstrated inExamples 16-19, were chosen for the study of dose-dependent targetknockdown in transgenic mice containing the human transthyretin gene.ISIS 420950 and ISIS 420955, which demonstrated 90% or more targetknockdown, but which also demonstrated toxicity in CD1 mice (Examples16-19) were also chosen for this study for comparison.

Treatment

Groups of four mice, two male and two female, each were injectedsubcutaneously twice a week for 4 weeks with 4 mg/kg, 10 mg/kg or 25mg/kg of ISIS 420913, ISIS 420921, ISIS 420922, ISIS 420950, ISIS420955, ISIS 420957, or ISIS 420959. One group of four mice, two maleand two female, was injected subcutaneously twice a week for 4 weekswith 25 mg/kg of the control oligonucleotide, ISIS 141923. One controlgroup of four mice, two male and two female, was injected subcutaneouslytwice a week for 4 weeks with PBS. Plasma samples were taken from eachgroup at days 0, 14 and 28. Two days after the last dose, the mice wereeuthanized and organs were harvested for further analysis.

RNA Analysis

RNA was extracted from liver tissue for real-time PCR analysis oftransthyretin using primer probe set RTS3029. Results are presented aspercent inhibition of human transthyretin, relative to PBS control. Asshown in Table 62, treatment with ISIS antisense oligonucleotidesresulted in significant dose-dependent reduction of human transthyretinmRNA in comparison to the PBS control. Treatment with the controloligonucleotide, ISIS 141923 did not result in significant reduction oftransthyretin, as expected.

TABLE 62 Inhibition of human transthyretin mRNA in the hTTR transgenicmice liver relative to the PBS control ISIS NO. Dose (mg/kg) %inhibition 420913 25 78 10 65 4 32 420921 25 76 10 64 4 13 420922 25 8010 53 4 21 420950 25 92 10 77 4 57 420955 25 88 10 56 4 23 420957 25 8510 72 4 32 420959 25 75 10 26 4 11 141923 25 0

Protein Analysis

Human transthyretin protein levels were measured in transgenic miceplasma by ELISA using an anti-transthyretin transthyretin polyclonalantibody (Abcam Ab37774) and a sheep anti-TTR horse radish peroxidasedetection antibody (Abcam cat. no. 35217). The color reaction wasdeveloped by the ImmunoPure® TMB Substrate Kit and absorbance measuredat 450 nm using a microtiter plate spectrophotometer. Plasma sampleswere taken predose and on days 7, 14, 21 and 28. The results arepresented in Table 63 expressed as percentage inhibition compared to thepredose levels and demonstrate a time-dependent and dose-dependentreduction in protein levels on treatment with ISIS oligonucleotides.

TABLE 63 Inhibition of human transthyretin protein in the hTTRtransgenic mice plasma relative to predose levels ISIS NO. Dose (mg/kg)d 0 d 14 d 28 420913 25 0 73 93 10 0 27 96 4 0 25 54 420921 25 0 73 9010 0 63 79 4 0 42 67 420922 25 0 63 96 10 0 57 89 4 0 38 77 420950 25 095 97 10 0 71 96 4 0 29 53 420955 25 0 84 96 10 0 53 91 4 0 20 30 42095725 0 83 93 10 0 51 66 4 0 32 49 420959 25 0 74 80 10 0 31 58 4 0 0 0141923 25 0 22 0

Body Weight and Organ Weight

The body weights of the mice were measured pre-dose and at the end ofthe treatment period. The body weights are presented in Table 64 and areexpressed as percent increase over the PBS control weight taken beforethe start of treatment. Liver, spleen and kidney weights were measuredat the end of the study, and are also presented in Table 64 as apercentage change over the respective organ weights of the PBS control.

TABLE 64 Change in body and organ weights of transgenic mice afterantisense oligonucleotide treatment (%) Body weight Liver Spleen KidneyPBS 6.4 0.0 0.0 0.0 ISIS 25 8.1 0.3 11.4 4.1 420913 10 10.6 −8.6 14.313.6 4 7.4 3.7 5.0 12.0 ISIS 25 10.5 8.8 25.6 −0.1 420921 10 9.7 5.710.8 4.0 4 8.7 −4.4 16.0 11.0 ISIS 25 8.4 5.6 18.0 1.7 420922 10 9.2−1.7 27.1 6.3 4 8.1 −2.1 −11.4 5.1 ISIS 25 12.8 14.3 22.8 1.7 420950 108.4 4.3 −2.8 0.6 4 9.1 0.4 14.2 1.5 ISIS 25 10.1 14.6 17.7 −4.4 42095510 11.8 5.6 −0.3 1.4 4 7.9 4.7 −12.3 4.5 ISIS 25 12.8 6.4 33.1 2.8420957 10 14.5 13.9 −6.3 9.7 4 7.4 −5.4 12.2 6.2 ISIS 25 10.0 2.4 72.723.3 420959 10 7.2 −5.4 40.2 9.8 4 4.1 −4.4 27.8 −6.6 ISIS 25 9.2 −1.320.4 −5.5 141923

Liver Function

To evaluate the effect of ISIS oligonucleotides on hepatic function,plasma concentrations of transaminases were measured using an automatedclinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.).Plasma concentrations of ALT (alanine transaminase) and AST (aspartatetransaminase) were measured and the results are presented in Table 65expressed in IU/L. Plasma levels of bilirubin were also measured usingthe same clinical chemistry analyzer; results are also presented inTable 65 and expressed in mg/dL.

TABLE 65 Effect of antisense oligonucleotide treatment on metabolicmarkers in the liver of transgenic mice Dose ALT AST TBIL (mg/kg) (IU/L)(IU/L) (mg/dL) PBS 47 63 0.16 ISIS 25 42 69 0.13 420913 10 49 90 0.17 442 59 0.18 ISIS 25 56 96 0.12 420921 10 51 68 0.22 4 42 75 0.14 ISIS 2550 76 0.12 420922 10 40 170 0.14 4 37 48 0.13 ISIS 25 74 116 0.14 42095010 37 67 0.13 4 34 64 0.11 ISIS 25 46 117 0.15 420955 10 54 76 0.16 4 50153 0.17 ISIS 25 40 73 0.13 420957 10 36 63 0.20 4 37 61 0.12 ISIS 25 5192 0.19 420959 10 48 69 0.13 4 37 67 0.13 ISIS 25 44 79 0.12 141923

Kidney Function

To evaluate the effect of ISIS oligonucleotides on kidney function,plasma concentrations of blood urea nitrogen (BUN) were measured usingan automated clinical chemistry analyzer (Hitachi Olympus AU400e,Melville, N.Y.). Results are presented in Table 66, expressed in mg/dL.

TABLE 66 Effect of antisense oligonucleotide treatment on BUN (mg/dL) inthe kidney of transgenic mice Dose (mg/kg) BUN PBS — 23 ISIS 25 24420913 10 22 4 20 ISIS 25 24 420921 10 22 4 23 ISIS 25 23 420922 10 22 424 ISIS 25 22 420950 10 26 4 23 ISIS 25 23 420955 10 24 4 25 ISIS 25 20420957 10 22 4 20 ISIS 25 25 420959 10 22 4 22 ISIS 25 19 141923

Example 21 Dose Response Confirmation of Antisense OligonucleotidesTargeting Human Transthyretin in Cynomolgus Monkey Primary Hepatocytes

Gapmers showing tolerability in CD1 mice and Sprague Dawley rats(studies described in Examples 17-19) as well as potency in transgenicmice (studies described in Examples 16 and 20) were selected and testedat various doses in primary hepatocytes of cynomolgus monkeys. Cellswere plated at a density of 35,000 cells per well and transfected usingelectroporation with 156.25 nM, 312.5 nM, 625 nM, 1,250 nM 2,500 nM,5,000 nM, 10,000 nM and 20,000 nM concentrations of antisenseoligonucleotide, as specified in Table 67. After a treatment period ofapproximately 16 hours, RNA was isolated from the cells andtransthyretin mRNA levels were measured by quantitative real-time PCR.Human transthyretin primer probe set RTS1396 was used to measure mRNAlevels. Transthyretin mRNA levels were adjusted according to total RNAcontent, as measured by RIBOGREEN®. Results are presented as percentinhibition of transthyretin, relative to untreated control cells. Asillustrated in Table 67, transthyretin mRNA levels were reduced in adose-dependent manner in hepatocytes treated with all the ISISoligonucleotides, which are cross-reactive with rhesus monkeytransthyretin gene, designated herein as SEQ ID NO: 4 (exons 1-4extracted from GENBANK Accession No. NW_001105671.1).

TABLE 67 Dose-dependent antisense inhibition of human transthyretin incynomolgus monkey primary hepatocytes using electroporation Target ISIS156.25 312.5 625 1250 2500 5000 10000 20000 IC₅₀ Start No. nM nM nM nMnM nM nM nM (μM) Site 304299 0 0 25 42 89 95 98 99 1.4 504 420913 0 0 4249 84 96 98 98 1.2 502 420915 0 8 46 58 84 94 97 99 1 505 420921 0 0 2630 53 74 94 97 2 512 420922 4 0 13 29 38 69 87 97 2.9 513 420950 23 2760 71 88 94 98 98 0.6 577 420955 19 0 25 50 74 86 93 97 1.4 582 420957 00 15 34 65 72 87 94 2.2 584 420959 3 12 10 37 71 88 94 94 1.5 586

Example 22 Measurement of Viscosity of ISIS Antisense OligonucleotidesTargeting Human Transthyretin

The viscosity of antisense oligonucleotides from studies described inExample 21 was measured with the aim of screening out antisenseoligonucleotides which have a viscosity more than 40 cP.Oligonucleotides having a viscosity greater than 40 cP would be tooviscous to be administered to any subject.

ISIS oligonucleotides (32-35 mg) were weighed into a glass vial, 120 μLof water was added and the antisense oligonucleotide was dissolved intosolution by heating the vial at 50° C. Part of (75 μL) the pre-heatedsample was pipetted to a micro-viscometer (Cambridge). The temperatureof the micro-viscometter was set to 25° C. and the viscosity of thesample was measured. Another part (20 μL) of the pre-heated sample waspipetted into 10 mL of water for UV reading at 260 nM at 85° C. (Cary UVinstrument). The results are presented in Table 68 and indicate that allthe antisense oligonucleotides solutions are optimal in their viscosityunder the criterion stated above.

TABLE 68 Viscosity and concentration of ISIS antisense oligonucleotidestargeting human transthyretin ISIS No. Viscosity (cP) Concentration(mg/mL) 304299 9.9 169 420913 6.5 178 420915 8.4 227 420921 8.2 234420922 5.3 191 420950 12.5 297 420955 15.7 259 420957 12.9 233 42095918.7 276

Example 23 Measurement of Half-Life of Antisense Oligonucleotide in CD1Mouse Liver

CD1 mice were treated with ISIS antisense oligonucleotides from studiesdescribed in Example 22 and the oligonucleotide half-life as well as theelapsed time for oligonucleotide degradation and elimination from theliver was evaluated.

Treatment

Groups of twelve CD1 mice each were injected subcutaneously twice perweek for 2 weeks with 50 mg/kg of ISIS 420913, ISIS 420921, ISIS 420922,ISIS 420950, ISIS 420955, ISIS 420957, and ISIS 420959. Four mice fromeach group were sacrificed 3 days, 28 days and 56 days following thefinal dose. Livers were harvested for analysis.

Measurement of Oligonucleotide Concentration

The concentration of the full-length oligonucleotide as well as thetotal oligonucleotide concentration (including the degraded form) wasmeasured. The method used is a modification of previously publishedmethods (Leeds et al., 1996; Geary et al., 1999) which consist of aphenol-chloroform (liquid-liquid) extraction followed by a solid phaseextraction. An internal standard (ISIS 355868, a 27-mer2′-O-methoxyethyl modified phosphorothioate oligonucleotide,GCGTTTGCTCTTCTTCTTGCGTTTTTT, designated herein as SEQ ID NO: 166) wasadded prior to extraction. Tissue sample concentrations were calculatedusing calibration curves, with a lower limit of quantitation (LLOQ) ofapproximately 1.14 μg/g. Half-lives were then calculated using WinNonlinsoftware (PHARSIGHT).

The results are presented in Tables 69, expressed as μg/g liver tissue.The half-life of each oligonucleotide is presented in Table 70.

TABLE 69 Full-length oligonucleotide concentration (μg/g) in the liverof CD1 mice ISIS No. 3 days 28 days 56 days 420913 243 109 33 420921 22549 6 420922 310 129 53 420950 254 88 62 420955 308 137 79 420957 325 12949 420959 258 97 37

TABLE 70 Half-life of oligonucleotide (days) in the liver of CD1 miceISIS No. Half-life (days) 420913 18.5 420921 10.0 420922 20.7 42095026.4 420955 27.2 420957 19.5 420959 18.9

Example 24 Effect of ISIS Antisense Oligonucleotides Targeting HumanTransthyretin in Cynomolgus Monkeys

Cynomolgus monkeys were treated with ISIS antisense oligonucleotidesfrom studies described in Examples 21, 22 and 23. Antisenseoligonucleotide efficacy and tolerability, as well as theirpharmacokinetic profile in the liver and kidney, were evaluated.

Treatment

Prior to the study, the monkeys were kept in quarantine for a 30-daytime period, during which standard panels of serum chemistry andhematology, examination of fecal samples for ova and parasites, and atuberculosis test, were conducted to screen out abnormal or ailingmonkeys. Nine groups of four randomly assigned male cynomolgus monkeyseach were injected subcutaneously thrice per week for the first week,and subsequently twice a week for the next 11 weeks, with 25 mg/kg ofISIS 304299, ISIS 420915, ISIS 420921, ISIS 420922, ISIS 420950, ISIS420955, ISIS 420957, or ISIS 420959. A control group of 4 cynomolgusmonkeys was injected with PBS subcutaneously thrice per week for thefirst week, and subsequently twice a week for the next 11 weeks. Bloodsamples were collected 5 days before the treatment as well as on variousdays of the study period and analyzed. The animals were fasted for atleast 13 hours (overnight) prior to blood collection. Terminalsacrifices of all groups were conducted on day 86, which was 48 hoursafter the last dose.

During the study period, the monkeys were observed daily for signs ofillness or distress. Any animal showing adverse effects to the treatmentwas removed and referred to the veterinarian and Study Director. All theanimals treated with ISIS 420955 were removed from the study on day 31due to symptoms of illness displayed by 2 monkeys in the group.Similarly, one monkey each from groups treated with ISIS 420957 and ISIS420950 was removed from the study on days 44 and 76, respectively, dueto signs of illness.

Inhibition Studies RNA Analysis

On day 86, RNA was extracted from liver tissue for real-time PCRanalysis of transthyretin using primer probe set RTS3029. Results arepresented as percent inhibition of transthyretin, relative to PBScontrol, normalized to cyclophilin. Similar results were obtained onnormalization with RIBOGREEN®. As shown in Table 71, treatment with ISISantisense oligonucleotides resulted in significant reduction oftransthyretin mRNA in comparison to the PBS control. Specifically,treatment with ISIS 420915 caused greater inhibition of TTR mRNA thantreatment with ISIS 304299, even though the two oligonucleotides differfrom each other by a single base-pair shift. The data for animalstreated with ISIS 420955 was taken at day 31.

TABLE 71 Inhibition of transthyretin mRNA in the cynomolgus monkey liverrelative to the PBS control ISIS No % inhibition 304299 59 420915 78420921 54 420922 61 420950 91  420955* 79 420957 64 420959 55 (*Data ofday 31)

Protein Analysis

The monkeys were fasted overnight prior to blood collection.Approximately 1 mL of blood was collected from all available animals andplaced in tubes containing the potassium salt of EDTA. The tubes werecentrifuged (3000 rpm for 10 min at room temperature) to obtain plasma.Transthyretin protein levels were measured in the plasma using aclinical analyzer. Plasma samples were taken predose (on day −5) and ondays 1, 9, 16, 23, 30, 44, 58, 72, and 86. The results are presented inTable 72 expressed as percentage inhibition compared to the predoselevels and demonstrate a time-dependent reduction in protein levels withtreatment with ISIS oligonucleotides. The final plasma TTR levels arepresented in Table 73 and demonstrate the strong correlation between TTRprotein level reduction and TTR mRNA inhibition (Table 71).Specifically, treatment with ISIS 420915 caused greater inhibition ofTTR plasma protein than treatment with ISIS 304299 (76% inhibition vs.47% inhibition), even though the two oligonucleotides differ from each

TABLE 72 Time course of transthyretin protein level reduction in thecynomolgus monkey plasma relative to predose levels ISIS Day Day Day DayDay Day Day Day Day No. 0 9 16 23 30 44 58 72 86 304299 4 15 21 23 26 2731 38 47 420915 2 8 23 34 42 54 63 70 76 420921 5 11 21 31 23 27 30 4050 420922 0 17 37 42 49 49 50 49 54 420950 0 39 63 68 72 79 85 82 87420955 0 42 63 80 81 n/a n/a n/a n/a 420957 2 18 28 26 26 35 35 41 50420959 0 25 29 28 32 38 42 43 50 n/a = study was terminated on day 31for animals treated with ISIS 420955; therefore data for subsequent daysis not available.

TABLE 73 Day 86 transthyretin protein level reduction in the cynomolgusmonkey plasma relative to predose levels ISIS No. % reduction 304299 47420915 76 420921 50 420922 54 420950 87 420957 50 420959 50

RBP4 protein levels were also measured in the plasma using an ELISA kit.Plasma samples were taken predose (on day −5) and on days 9, 16, 23, 30,44, 58, 72, and 86. The results are presented in Table 74 expressed aspercentage inhibition compared to the predose levels. Some of the ISISoligonucleotides (ISIS 420915, ISIS 420922, ISIS 420950, ISIS 420955 andISIS 420959) demonstrate a time-dependent reduction in protein levels,concomitant with TTR reduction. The final plasma RBP4 levels arepresented in Table 75 and also demonstrate the strong correlationbetween RBP4 and TTR protein level reductions (Table 73) on treatmentwith the above-mentioned oligonucleotides. Specifically, treatment withISIS 420915 caused greater inhibition of RBP4 plasma protein thantreatment with ISIS 304299 (63% inhibition vs. 19% inhibition), eventhough the two oligonucleotides differ from each other by a singlebase-pair shift.

TABLE 74 Time course of RBP4 protein level reduction in the cynomolgusmonkey plasma relative to predose levels ISIS Day Day Day Day Day DayDay Day No. 9 16 23 30 44 58 72 86 304299 0 6 10 4 1 9 13 19 420915 5 2222 30 38 47 54 63 420921 0 0 0 0 0 0 6 24 420922 4 19 16 34 33 29 15 32420950 30 44 46 47 52 54 47 48 420955 6 36 53 65 n/a n/a n/a n/a 4209570 10 0 0 0 0 3 27 420959 18 22 14 17 19 25 22 34 n/a = study wasterminated on day 31 for animals treated with ISIS 420955; thereforedata for subsequent days is not available.

TABLE 75 Day 86 RBP4 protein level reduction in the cynomolgus monkeyplasma relative to predose levels ISIS No. % reduction 304299 19 42091563 420921 24 420922 32 420950 48 420957 27 420959 34

Tolerability Studies Body and Organ Weight Measurements

To evaluate the effect of ISIS oligonucleotides on the overall health ofthe animals, body and organ weights were measured at day 86. The datafor animals treated with ISIS 420955 was taken at day 31. Body weightswere measured and compared to that at pre-dose levels. Organ weightswere measured and treatment group weights were compared to thecorresponding PBS control weights. The data is presented in Table 76.

TABLE 76 Final body and organ weight % changes in the cynomolgus monkeyrelative to predose levels Body Liver Kidney Spleen ISIS No. weightweight weight weight 304299 +6 +27 +37 +53 420915 +6 +37 +26 +41 420921+4 +42 +43 +22 420922 +4 +45 +39 +63 420950 0 +204 +166 +297  420955* −3+36 +81 +70 420957 −6 +55 +184 +109 420959 0 +57 +101 +112 (*Data of day31)

Liver Function

To evaluate the effect of ISIS oligonucleotides on hepatic function,blood samples were collected from all the study groups. The bloodsamples were collected in tubes without any anticoagulant for serumseparation. The tubes were kept at room temperature for 90 min and thencentrifuged (3000 rpm for 10 min at room temperature) to obtain serum.Concentrations of transaminases were measured using a Toshiba 200FR NEOchemistry analyzer (Toshiba Co., Japan). Plasma concentrations of ALT(alanine transaminase) and AST (aspartate transaminase) were measured onday 86 and the results are presented in Table 77, expressed in IU/L.Alkaline phosphatase, which is synthesized in increased amounts bydamaged liver cells, is also a marker of liver disease and was similarlymeasured. C-reactive protein (CRP), which is synthesized in the liverand which serves as a marker of inflammation, was also similarlymeasured on day 86. Both alkaline phosphatase and CRP data are alsopresented in Table 77. Bilirubin is also a liver metabolic marker andwas similarly measured and is presented in Table 77, expressed in mg/dL.

TABLE 77 Effect of antisense oligonucleotide treatment on livermetabolic markers in cynomolgus monkey plasma AST ALT ALP CRP Bilirubin(IU/L) (IU/L) (IU/L) (mg/L) (mg/dL) PBS 60 54 955 2.4 0.24 ISIS 30429981 101 747 3.3 0.17 ISIS 420915 68 62 672 1.6 0.15 ISIS 420921 98 107832 3.2 0.14 ISIS 420922 94 96 907 2.4 0.15 ISIS 420950 132 94 1032 12.90.11 ISIS 420957 100 73 868 23.5 0.15 ISIS 420959 70 63 811 16.0 0.13

Kidney Function

To evaluate the effect of ISIS oligonucleotides on kidney function,blood samples were collected from all the study groups. The bloodsamples were collected in tubes without any anticoagulant for serumseparation. The tubes were kept at room temperature for 90 min and thencentrifuged (3000 rpm for 10 min at room temperature) to obtain serum.Concentrations of BUN and creatinine were measured at day 86 using aToshiba 200FR NEO chemistry analyzer (Toshiba Co., Japan). Results arepresented in Table 78, expressed in mg/dL.

Urine samples were collected by drainage from special stainless-steelcage pans on day 5 before the study, and subsequently on days 25 and 84.Urine total protein to creatinine ratio was measured using a Toshiba200FR NEO chemistry analyzer (Toshiba Co., Japan) and the results arepresented in Table 79.

TABLE 78 Effect of antisense oligonucleotide treatment on plasma BUN andcreatinine levels (mg/dL) in cynomolgus monkeys BUN Creatinine PBS 280.86 ISIS 304299 27 0.85 ISIS 420915 25 0.90 ISIS 420921 33 0.99 ISIS420922 28 0.86 ISIS 420950 36 0.97 ISIS 420957 35 0.86 ISIS 420959 270.89

TABLE 79 Effect of antisense oligonucleotide treatment on urine proteinto creatine ratio in cynomolgus monkeys Day −5 Day 25 Day 84 PBS 0.0030.01 0.00 ISIS 304299 0.000 0.01 0.00 ISIS 420915 0.003 0.00 0.00 ISIS420921 0.033 0.13 0.09 ISIS 420922 0.010 0.05 0.02 ISIS 420950 0.0080.29 0.21 ISIS 420955 0.000 0.61 n/a ISIS 420957 0.000 0.48 0.36 ISIS420959 0.005 0.08 0.03 n/a = study was terminated on day 31 for animalstreated with ISIS 420955; therefore data for subsequent days is notavailable.

Hematology

To evaluate any inflammatory effect of ISIS oligonucleotides incynomolgus monkeys, blood samples were approximately 0.5 mL of blood wascollected from each of the available study animals in tubes containingthe potassium salt of EDTA. Samples were analyzed for red blood cell(RBC) count, white blood cells (WBC) count, individual white blood cellpercentages, such as that of monocytes, neutrophils, lymphocytes, aswell as for platelet count and hematocrit (%), using an ADVIA120hematology analyzer (Bayer, USA). The data is presented in Table 80.

TABLE 80 Effect of antisense oligonucleotide treatment on hematologicparameters in cynomolgus monkeys WBC RBC Platelet Hematocrit LymphocytesNeutrophil Monocytes (×10³/μL) (×10⁶/μL) (×1000/μL) (%) (%) (%) (%) PBS9.6 5.3 415 40 62 35 1.8 ISIS 304299 11.6 5.2 395 38 68 26 3.1 ISIS420915 10.3 5.1 382 36 72 22 3.5 ISIS 420921 9.8 5.2 385 36 60 34 2.5ISIS 420922 11.6 5.2 396 37 62 29 5.4 ISIS 420950 13.7 4.4 260 33 51 347.8 ISIS 420957 18.6 4.7 298 33 52 35 9.1 ISIS 420959 7.7 4.8 306 32 6229 5.5

Analysis of Factors of Inflammation

To evaluate the effect of ISIS oligonucleotides on factors involved ininflammation, blood was collected on day 86 from all available animalsfor complement C3 analysis, as well as for measurement of cytokinelevels. For complement C3 analysis, the blood samples were collected intubes without anticoagulant for serum separation. The tubes were kept atroom temperature for 90 min and then centrifuged (3000 rpm for 10 min atroom temperature) to obtain serum. Complement C3 was measured using anautomatic analyzer (Toshiba 200 FR NEO chemistry analyzer, Toshiba co.,Japan). The data is presented in Table 81, expressed in mg/dL.

For cytokine level analyses, blood was collected in tubes containingEDTA for plasma separation. The tubes were then centrifuged (3000 rpmfor 10 min at room temperature) to obtain plasma. Plasma samples weresent to Aushon Biosystems Inc. (Billerica, Mass.) for measurement ofchemokine and cytokine levels. Levels of TNF-α were measured using therespective primate antibodies and levels of MIP-1α, MCP-1, and MIP-1βwere measured using the respective cross-reacting human antibodies.Measurements were taken 5 days before the start of treatment and on days3 and 86. The results are presented in Tables 82-85.

TABLE 81 Effect of antisense oligonucleotide treatment on complement C3(mg/dL) in cynomolgus monkeys C3 PBS 133 ISIS 304299 96 ISIS 420915 104ISIS 420921 91 ISIS 420922 102 ISIS 420950 70 ISIS 420957 69 ISIS 42095995

TABLE 82 Effect of antisense oligonucleotide treatment on MCP-1 (pg/mL)in cynomolgus monkeys Day −5 Day 3 Day 86 PBS 232 362 206 ISIS 304299219 292 427 ISIS 420915 204 342 400 ISIS 420921 281 407 2120 ISIS 420922215 482 838 ISIS 420950 170 370 3355 ISIS 420957 208 308 3485 ISIS420959 237 715 2035

TABLE 83 Effect of antisense oligonucleotide treatment on TNF-α (pg/mL)in cynomolgus monkeys Day −5 Day 3 Day 86 PBS 60 46 16 ISIS 304299 46 3524 ISIS 420915 113 83 30 ISIS 420921 57 50 56 ISIS 420922 30 59 46 ISIS420950 48 54 266 ISIS 420957 29 33 87 ISIS 420959 22 77 74

TABLE 84 Effect of antisense oligonucleotide treatment on MIP-1α (pg/mL)in cynomolgus monkeys Day −5 Day 3 Day 86 PBS 6 7 7 ISIS 304299 6 7 9ISIS 420915 5 5 10 ISIS 420921 8 11 9 ISIS 420922 9 8 5 ISIS 420950 7 95 ISIS 420957 6 6 6 ISIS 420959 9 6 5

TABLE 85 Effect of antisense oligonucleotide treatment on MIP-1β (pg/mL)in cynomolgus monkeys Day −5 Day 3 Day 86 PBS 13 19 42 ISIS 304299 17 2354 ISIS 420915 15 27 72 ISIS 420921 23 43 112 ISIS 420922 9 41 70 ISIS420950 8 25 126 ISIS 420957 16 27 182 ISIS 420959 36 46 117

Coagulation Tests

To evaluate the effect of ISIS oligonucleotides on factors involved inthe coagulation pathway, the standard tests for coagulation wereemployed. PT and aPTT were measured using platelet poor plasma (PPP)from the monkeys over a period of 48 hrs. PT and aPTT values areprovided in Tables 86 and 87 and expressed in seconds. Fibrinogen levelson the plasma were also quantitated over a period of 48 hrs and the datais presented in Table 88. As shown in Tables 86-88, PT, aPTT andfibrinogen were not significantly altered in monkeys treated with ISISoligonucleotides compared to the PBS control.

TABLE 86 Effect of antisense oligonucleotide treatment on PT (sec) 0 hr1 hr 4 hr 8 hr 24 hr 48 hr PBS 10.08 10.38 10.10 10.33 9.83 9.40 ISIS304299 10.38 10.30 10.48 10.20 9.95 9.53 ISIS 420915 10.15 10.13 10.389.93 9.75 9.48 ISIS 420921 10.28 10.13 10.43 10.18 9.80 9.55 ISIS 4209229.95 10.00 10.05 9.70 9.48 9.28 ISIS 420950 10.30 10.47 10.57 10.27 9.639.50 ISIS 420957 10.63 10.47 10.60 10.77 10.33 10.27 ISIS 420959 10.0810.10 10.20 10.15 9.80 9.55

TABLE 87 Effect of antisense oligonucleotide treatment on aPTT (sec) 0hr 1 hr 4 hr 8 hr 24 hr 48 hr PBS 19.40 19.70 20.13 20.20 19.43 17.30ISIS 304299 21.83 24.35 27.05 25.73 22.40 18.78 ISIS 420915 20.05 22.8323.83 24.00 21.78 17.90 ISIS 420921 24.15 26.68 31.78 31.90 27.80 22.15ISIS 420922 25.28 29.48 34.83 33.90 29.13 25.08 ISIS 420950 28.13 31.4035.40 35.40 31.40 28.37 ISIS 420957 29.13 33.27 39.13 37.40 36.50 29.93ISIS 420959 22.45 24.73 29.18 28.38 25.50 20.65

TABLE 88 Effect of antisense oligonucleotide treatment on fibrinogen(mg/dL) 0 hr 1 hr 4 hr 8 hr 24 hr 48 hr PBS 212 203 240 247 282 272 ISIS304299 175 172 198 207 227 200 ISIS 420915 213 196 204 258 257 215 ISIS420921 208 209 230 237 301 249 ISIS 420922 278 277 335 338 400 304 ISIS420950 293 295 348 376 390 296 ISIS 420957 280 299 344 330 434 328 ISIS420959 276 277 354 326 414 320

Thyroid Panel Analysis

To evaluate the effect of ISIS oligonucleotides on thyroid hormones,monkeys were fasted overnight and 3.5 mL of blood was drawn from each ofthe available study animals 5 days prior to the start of treatment andon days 51 and 86. Collected blood samples were kept in tubes withoutanticoagulant for serum separation. The tubes were kept for 90 min atroom temperature, after which they were centrifuged (3000 rpm for 10 minat room temperature) to obtain serum. Serum samples were sent to theBiomarkers Core Laboratory of Emory University (Atlanta, Ga.) forthyroid panel analysis. The results for thyroid stimulating hormone(TSH) are provided in Table 89 and expressed μL/mL. The results fortotal and free T3 hormone are provided in Tables 90 and 91.The resultsfor total and free T4 hormone are provided in Tables 92 and 93. Overall,the thyroid panel analysis showed that all the animals remained withinacceptable hormone levels even though transthyretin expression levelswere reduced, demonstrating that the transthyretin antisenseoligonucleotides did not affect hormone levels.

TABLE 89 Effect of antisense oligonucleotide treatment on TSH (μL/mL)Day −5 Day 51 Day 86 PBS 0.8 0.7 1.0 ISIS 304299 1.4 1.0 2.2 ISIS 4209151.4 1.5 2.5 ISIS 420921 0.7 0.6 1.0 ISIS 420922 1.0 1.2 1.9 ISIS 4209500.6 2.2 5.4 ISIS 420957 0.6 2.6 4.9 ISIS 420959 0.9 1.6 4.7

TABLE 90 Effect of antisense oligonucleotide treatment on total T3(ng/dL) Day −5 Day 51 Day 86 PBS 177 248 140 ISIS 304299 202 226 176ISIS 420915 156 206 156 ISIS 420921 217 204 137 ISIS 420922 188 177 131ISIS 420950 260 208 105 ISIS 420957 266 160 78 ISIS 420959 299 219 137

TABLE 91 Effect of antisense oligonucleotide treatment on free T3(pg/mL) Day −5 Day 51 Day 86 PBS 7.7 5.8 5.2 ISIS 304299 9.2 6.0 4.7ISIS 420915 8.9 5.6 4.5 ISIS 420921 10.2 4.8 4.0 ISIS 420922 8.9 5.4 3.7ISIS 420950 7.2 3.8 2.1 ISIS 420957 8.8 4.0 2.4 ISIS 420959 8.3 4.9 3.3

TABLE 92 Effect of antisense oligonucleotide treatment on total T4(ng/dL) Day −5 Day 51 Day 86 PBS 5.8 4.9 4.4 ISIS 304299 8.1 5.5 6.1ISIS 420915 8.3 5.7 5.5 ISIS 420921 7.6 6.1 5.6 ISIS 420922 7.3 6.1 5.8ISIS 420950 6.1 6.3 5.7 ISIS 420957 6.3 4.4 5.0 ISIS 420959 7.9 5.9 8.1

TABLE 93 Effect of antisense oligonucleotide treatment on free T4(pg/mL) Day −5 Day 51 Day 86 PBS 3.4 2.4 1.7 ISIS 304299 3.2 2.5 1.7ISIS 420915 5.0 1.8 1.7 ISIS 420921 2.6 1.5 1.5 ISIS 420922 3.5 1.6 1.5ISIS 420950 2.5 1.2 1.1 ISIS 420957 2.4 1.2 1.2 ISIS 420959 3.8 1.4 1.5

Pharmacokinetic Studies Measurement of Oligonucleotide Concentration

The concentration of the full-length oligonucleotide as well as thetotal oligonucleotide concentration (including the degraded form) wasmeasured. The method used is a modification of previously publishedmethods (Leeds et al., 1996; Geary et al., 1999) which consist of aphenol-chloroform (liquid-liquid) extraction followed by a solid phaseextraction. An internal standard (ISIS 355868, a 27-mer2′-O-methoxyethyl modified phosphorothioate oligonucleotide,GCGTTTGCTCTTCTTCTTGCGTTTTTT, designated herein as SEQ ID NO: 166) wasadded prior to extraction. Tissue sample concentrations were calculatedusing calibration curves, with a lower limit of quantitation (LLOQ) ofapproximately 1.14 μg/g. The ratio of the concentrations in the kidneyversus the liver was calculated. The results are presented in Tables 94and 95, expressed as μg/g tissue.

TABLE 94 Full-length oligonucleotide concentration (μg/g) in the liverof cynomolgus monkey Kidney/Liver ISIS No. Kidney Liver ratio 3042992179 739 2.9 420915 2439 1064 2.3 420921 4617 1521 3.0 420922 3957 11263.5 420950 3921 1082 3.6 420955 2444 1111 2.2 420957 3619 1230 2.9420959 3918 1158 3.4

TABLE 95 Total oligonucleotide concentration (μg/g) in the liver ofcynomolgus monkey Kidney/Liver ISIS No. Kidney Liver ratio 304299 3098992 3.1 420915 3024 1266 2.4 420921 6100 1974 3.1 420922 4861 1411 3.4420950 6003 1553 3.9 420955 2763 1208 2.3 420957 5420 1582 3.4 4209595498 1501 3.7

1-40. (canceled)
 41. A compound consisting of a conjugate group and asodium salt of a single-stranded modified oligonucleotide consisting of20 linked nucleosides having a nucleobase sequence consisting of SEQ IDNO: 80, wherein the modified oligonucleotide comprises: a gap segmentconsisting of ten linked deoxynucleosides; a 5′ wing segment consistingof five linked nucleosides; and a 3′ wing segment consisting of fivelinked nucleosides; wherein the gap segment is positioned between the 5′wing segment and the 3′ wing segment, wherein each nucleoside of eachwing segment comprises a 2′-O-methoxyethyl sugar, wherein eachinternucleoside linkage is a phosphorothioate linkage, and wherein eachcytosine of the modified oligonucleotide is a 5-methylcytosine.
 42. Acomposition comprising the compound of claim
 41. 43. A method oftreating transthyretin amyloidosis in an individual comprisingadministering to the individual the compound of claim
 41. 44. The methodof claim 43, wherein the transthyretin amyloidosis is familial amyloidpolyneuropathy (FAP).
 45. The method of claim 43, wherein thetransthyretin amyloidosis is familial amyloid cardiopathy (FAC).
 46. Amethod of treating transthyretin amyloidosis in an individual comprisingadministering to the individual the composition of claim
 42. 47. Themethod of claim 46, wherein the transthyretin amyloidosis is familialamyloid polyneuropathy (FAP).
 48. The method of claim 46, wherein thetransthyretin amyloidosis is familial amyloid cardiopathy (FAC).